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TABLE OF CONTENTS 1.0 INFLAMMATION 1.1 Purification of mononuclear and polymorphonuclear cells............... 4 1.2 Isolation of cells from the peritoneal cavities of mice....................... 8 1.3 Adherence purification of monocytes............................................... 9 1.4 Antibody- or C3b-dependent phagocytosis...................................... 10 1.5 Activation of macrophages .............................................................. 12 1.6 Monokine Bioassays........................................................................ 14 1.6.1 Assay for IL-1 activity......................................................... 14 1.6.2 Assay for IL-6 activity......................................................... 17 1.6.3 Cytotoxicity assay for TNFα............................................... 19 1.7. Neutrophil chemotaxis assay.......................................................... 21 1.8. FcεRI-dependent activation of mast cells ....................................... 24 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION 2.1 Hybridoma cell culture with production of monoclonal antibodies.... 26 2.2 Affinity purification of IgG antibodies ............................................... 27 2.2.1 Avid-AL affinity chromatography ........................................ 27 2.2.2 Protein A-Sepharose affinity chromatography ................... 29 2.3 Preparation of IgM antibodies.......................................................... 31 2.4 Analysis & characterization of immunoglobulins.............................. 33 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins..... 33 2.4.2 Western blotting to detect immunoglobulins ...................... 35 3.0 T CELL AND B CELL RESPONSES 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells ........................ 37 3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells .......... 39 3.3 Assessment of T cell proliferation.................................................... 41 3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells .............. 44 3.5 ELISPOT assays for single cytokine- or Ab-producing cells ............ 45 3.6 ELISA assay for detection of antigen-specific antibodies ................ 49 3.7 In vivo assessment of T cell responses ........................................... 51 3.8 Immunohistochemical detection of cytokines in tissues................... 53 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION 4.1 Northern blotting ........................................................................... 55 4.1.1 Purification of cellular RNA ................................................ 55 4.1.2 Electrophoresis of RNA & transfer to membranes ............. 58 4.1.3 32P-labelled cDNA probe synthesis ................................... 61 4.1.4 Pre-hybridization, hybridization, and washing .................... 63 4.1.5 Detection of mRNA bands ................................................. 65 4.2 In Situ hybridization ...................................................................... 67 4.2.1 Probe synthesis & purification............................................ 67 4.2.2 Preparation of slides for hybridization ................................ 71 4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA ... 74
4.2.4 Post-hybridization washing & autoradiography .................. 76 4.2.5 Autoradiograph development & counter-staining ............... 78 4.3 Semi-quantitative RT-PCR to detect cytokine mRNA ................. 79 4.3.1 First strand cDNA Synthesis using Oligo(dT) priming ........ 80 4.3.2 PCR amplification of the target cDNA ................................ 81 4.3.2 Detection of RT-PCR products .......................................... 82 APPENDICES 5.1 APPENDIX A -- GENERAL METHODS .......................................... 83 5.1.1 Anti-sheep RBC antisera ................................................... 83 5.1.2 Cell counting ...................................................................... 83 5.1.3 C3b opsinization of yeast................................................... 84 5.1.4 Cytocentrifuge preparations............................................... 85 5.1.5 Dialysis tubing.................................................................... 85 5.1.6 Fixation of tissues for ISH or IHC....................................... 85 5.1.7 Lung cells (single cell suspension)..................................... 86 5.1.8 Lysis of red blood cells....................................................... 86 126.96.36.199 Hypotonic lysis with H2O...................................... 86 188.8.131.52 Lysis with ammonium chloride ............................. 86 5.1.9 Opsinization of SRBC with antibody .................................. 87 5.4.10 Protein assay in microtiter plates ..................................... 87 5.1.11 Splenocytes (single cell suspensions) ............................. 87 5.1.12 Splenocytes (spleen cell-conditioned medium) ................ 88 5.1.13 Staining Protocols ............................................................ 89 184.108.40.206 Giemsa stains .................................................... 89 220.127.116.11.1 Wrights-Giemsa staining ...................... 89 18.104.22.168.2 Giemsa staining of tissue sections ....... 89 22.214.171.124 Gills hematoxylin for IHC.................................... 90 126.96.36.199 Toluidine blue staining (ISH counter-stain)......... 90 5.1.14 Standard curves (e.g., cytokines) .................................... 90 5.1.15 TESPA-treatment of glass slides ..................................... 91 5.2 APPENDIX B -- REAGENTS & SOLUTIONS CELLULAR IMMUNOLOGY REAGENTS................................... 92 Acidified isopropanol ......................................................... 92 Actinomycin D ................................................................... 92 Alsevers solution ............................................................... 92 Ammonium chloride .......................................................... 92 Ammonium sulfate (saturated solutions) ........................... 92 Borate-buffered saline....................................................... 92 ELISPOT & ELISA Carbonate Coating buffer ................... 92 Isotonic Percoll Density Gradient Medium ........................ 93 PAGE running buffer ......................................................... 93 PAGE 2x sample prep buffer ............................................ 93 PAGE gel fix buffer............................................................ 93 Phosphate-buffered saline (PBS)...................................... 93
Giemsa Stain..................................................................... 93 Giemsa Stock Solution...................................................... 93 0.4% Trypan Blue.............................................................. 94 MOLECULAR BIOLOGY REAGENTS Agarose/formaldehyde/MOPS gel (for Northerns)............. 94 Cesium Chloride (for isolation of total cellular RNA) ......... 94 DEPC-treated water (& other solutions) ............................ 94 Dithiothreitol ...................................................................... 94 EDTA (0.5M) ..................................................................... 94 Guanidinium Isothiocyanate (GSCN) ............................... 94 ISH 10x salts ..................................................................... 94 ISH hybridization buffer..................................................... 95 Northern blotting pre-hyb/hybridization solution ................ 95 MOPS (1 M) ...................................................................... 95 5X MOPS Buffer................................................................ 95 Phenol (salt-saturated)...................................................... 95 Reagents for purifying DNA from agarose gels ................. 96 General purpose restriction endonuclease buffers............ 96 RNA sample prep buffer (Northern analysis) .................... 96 RNA sample dye/loading buffer......................................... 96 RNAse A ........................................................................... 96 Salmon sperm DNA .......................................................... 96 Sodium acetate (3M)......................................................... 96 20X SSC (4 liters) ............................................................. 97 STE buffer ......................................................................... 97 5.3 APPENDIX C -- TISSUE CULTURE MEDIA Click's medium............................................................................. 98 DMEM.......................................................................................... 98 DMEM-0% FCS ........................................................................... 98 DMEM-10% FCS. ........................................................................ 98 DMEM-10% normal horse serum................................................. 98 HBSS (Ca++ and Mg++-free) ...................................................... 98 MEM ............................................................................................ 98 RPMI 1640................................................................................... 98 RPMI-0% FCS. ............................................................................ 98 RPMI-10% FCS ........................................................................... 98 5.4 APPENDIX D -- MAINTENANCE OF CELL LINES 7TD1 cells (for assay of IL-6)....................................................... 99 Cl.MC/C57.1 cells (C57 mast cells) ............................................. 99 L-929 cells (for TNF bioassay)..................................................... 99 LM-1 cells (for assay of IL-1) ....................................................... 99 Pu5-1.8 cells (macrophage cell line) ............................................ 99 5.6 APPENDIX F -- Human cytokine RT-PCR primers......................... 101 5.7 APPENDIX G -- WWW Immunology sites of interest ...................... 102
5.7 APPENDIX G -- Selected templates for 96--well plates .................. 103
1.0 INFLAMMATION: In the first week of this course we will begin to acquire some of the basic skills needed to examine several components of the inflammatory cascade, a series of responses that are integrally intertwined with the immune system. First we will begin to acquire some basic skills in tissue culture, and the purification of selected cells associated with the immunoinflammatory system (i.e., peripheral blood [PBL] monocytes and neutrophils [PMN or polymorphonuclear cells]). Then we will learn how to identify them using morphologic criteria, and how to assess a couple of functions associated with monocytes/macrophages - phagocytosis (a subfunction of antibody-dependent cellular cytotoxicity) and activation-dependent monokine production. 1.1 Purification of mononuclear and polymorphonuclear cells from mouse peripheral blood. While this protocol is designed specifically for the purification of cells from the peripheral blood, such density gradient systems can also be used for other cell systems (e.g., to purify PMN from glycogen-elicited peritoneal cavity preparations). In general it is better to use polypropylene [p.p.] rather than polystyrene [p.s.] tubes because polypropylene is less "sticky" for the cells and therefore activates them to a lesser extent than polystryrene. On the other hand, polypropylene tubes are more expensive, so this too needs to be taken into account. Materials BALB/c mouse anaesthetic (methoxyfluorane) clinical centrifuge, 15 ml centrifuge tubes, 4 ml round bottom p.s. tubes 23-25 ga needles & 1 ml syringes pipettes/pipettors (micro- and macropipettes) microscope slides (frosted end; & pencil for labeling) sterile surgical tools (optional, for open body cardiac puncture) laminar flow hood, inverted microscope, hemocytometer Reagents anticoagulant (heparin 1000U/ml) DMEM with 1 U/ml heparin or with 3 mg/ml EDTA anticoagulant DMEM-10% FCS (see Appendix A)
70% ethanol isotonic Percoll(see Appendix C) RBC sedimentation buffer (4.5% dextran-T500 in PBS) cytocentrifuge METHOD 1. Obtain blood from surgically anaesthetized (or euthanized) mouse either by closed- or open-body cardiac puncture. Withdraw blood slowly into a 1 ml syringe containing 100 µl of heparin, being careful not to hemolyse the red blood cells by using too much back pressure on syringe. Work fairly quickly, or mix the blood with the anticoagulant in the syringe fairly often, so that the blood doesn't coagulate. 2. Mix the blood with the RBC sedimentation buffer (1 volume sed. buffer: 1 volume blood) in the 4 ml tubes and allow the mixture to stand undisturbed on the bench for 20 - 45' (the time can be highly variable, depending on the species of animal)1. The RBC's will form rouleaux (chains of RBC's) that will sediment out of suspension rather rapidly, leaving you with a leukocyte/platelet-rich plasma layer above the settled RBC's. 3. Withdraw the plasma layer and transfer to a 15 ml polypropylene tube. Add 10 12 ml of DMEM-anticoagulant to the plasma and sediment the leukocytes out of suspension by centrifugation (1000 rpm for 10')2,3. Aspirate the platelet-rich medium, avoiding the cell pellet, and then resuspend the cells by vigorously flicking the tube. When the cell pellet has become a 'paste' on the lower walls of the tube, add ≈5 - 10 ml of tissue culture medium and then gently vortex by hand to ensure that you have a 'single cell suspension' (i.e., no clumps of cells). 4. While the cells are spinning in step 3, prepare a 70% isotonic Percoll gradient cushion by mixing together 1.75 ml isotonic Percoll and 0.75 ml of
RBC do not sediment from bovine blood using this dextran sedimentation protocol. Alternately, with bovine blood, one can either use the density gradients directly with diluted (1:1 with PBS) anticoagulated blood, followed by RBC lysis of the red cell rich-PMN fraction, or use the buffy coat of white blood cells from the top of sedimented whole anticoagulated whole blood and fractionate these cells using density gradient centrifugation as noted. 2 This fairly low speed centrifugation step reduces the platelet contamination of the mononuclear cell fraction of the blood. Platelets are a very rich source of a number of cytokines (including TGFβ) and other potent biologically active mediators. 3 Alternately, the platelet-rich leukocyte-plasma can be loaded directly onto the gradients. This saves some time in the purification procedure, but leaves the mononuclear cell fraction containing high levels of platelets, which can be gotten rid of by a subsequent low-speed spin (i.e., 1000 rpm for 10 min, in the clinical centrifuge)
Centrifuge the cell gradients for ≈25 min at ≈2000 rpm (room temperature) in the clinical centrifuge. After resuspending the cell pellet from step 3. no brake). Transfer the cells to a new tube and dilute any of the density gradient medium by topping up the tube with additional medium. If the PMN are badly contaminated with red blood cells.e. LSM gradients are run for only 15-25 min. carefully aspirate the remaining Percoll solution. one can use other density gradient media. then resuspend the pelleted cells by flicking the tube and then adding a large volume of medium. The completed gradient will appear much as it did before spinning. tube4. . trying to avoid aspirating any substantial volume of the Percoll. Furthermore.. and allow the centrifuge rotor to coast to a stop (i. in a 15 ml p.p. gently layer this suspension on top of the 70% isotonic Percoll 'cushion'. To harvest the PMN. 70% C 4 Alternately. but now the PMN and RBC will reside as a pellet at the bottom of the tube and the mononuclear cells (monocytes and lymphocytes) will reside as a band of cells at the interface between the tissue culture medium and the isotonic Percoll. DMEM/heparin/EDTA.7 5. To harvest the mononuclear cells. which is useful for the preparation of mononuclear cells from an array of species. simply pipette the cells directly from the interface. such as Ficoll-Hypaque or Lymphocyte Separation Medium (see ALTERNATE PROTOCOL). so that there is no mixing of the two layers. these can by lysed by hypotonic lysis or ammonium chloride (see Appendix D).
rewash at low speed (i. The drawback to using an abbreviated centrifugation time is that neutrophils will not sediment to the bottom of the gradients in this time. 800 rpm for 10').. Obtain leukocyte/platelet-rich plasma as in the Percoll procedure (§1. 1 ml) of medium. one can use Lymphocyte Separation Medium (LSM. ovine). which seems to be useful across a wide array of species (e.30 min..g.. Organon-Technika Inc) METHOD 1-3. Remove 20 µl of each preparation for cell counting by hemocytometer and determine the cell numbers and viabilities (see Appendix D). bovine. but instead will remain suspended in and can be recovered from the LSM rather than as a pellet at the bottom of the tube. . set them up in culture at a cell density of 3x106 cells/ml and challenge both the monocytes and PMN with endotoxin as in §1. and resuspend the cells in a minimal volume (e. ≈400xg) for 15 . If you have sufficient cells remaining. murine. Wash both cell preparations (i. Prepare cytocentrifuge slides of the cells by applying 5x104 cells in ≈100 µl of medium to each slide assembly and centrifuging them for 5 min at 1500 rpm.8 6. 1. 7.e. with the exception that the cells are centrifuged on the gradients at 1500 rpm (i. PMN and mononuclear cells) as above..1).1) Reagents all reagents required for the Percoll isolation procedure (§1. Thus the spin is shorter and at a much lower speed (which may be desirable in terms of the harshness of cellular treatment as well as in term so the times involved). 8. Materials all materials required for the Percoll isolation procedure (§1. Organon-Technika)..e. human.1) Lymphocyte Separation Medium (LSM. equine. Allow the sedimented cells to dry well before staining them with Giemsa solution (see Appendix D).1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) Rather than using Percoll or Ficoll-Paque gradients to separate the mononuclear cells from the other leukocytes. If the mononuclear cell fraction is heavily contaminated with platelets.e. The procedures employed are essentially identical to those for the Percoll gradients.g.
step 5.9 4. 6 The formula for calculating the volumes of materials needed to achieve specific densities with Percoll is: .. and wash them as in §1. lymphocytes.e.e.25 min5 at 1500 rpm in the clinical centrifuge (i. then carefully overlay this with either the platelet-rich leukocyte-plasma or with platelet depleted. Harvest the mononuclear cells as a band from the top of the density gradient medium. These are relatively easy to generate as custom gradients which can be tailored for individual needs by simply changing the low and high density "ends" of the gradient. as in §1. Centrifuge the gradients for 15 . Pipette 3 ml of LSM into a 15 ml p. 25') is required to sediment these cells. TABLE . basophils. 5.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations) The individual leukocyte populations (i.1 . conical centrifuge tube. eosinophils and neutrophils) can be at least partially purified as discrete bands directly from continuous density gradients. 1.1) 5 Fifteen minutes is usually adequate to fully purify the mononuclear cells from the polymorphonuclear cells and is sufficient to sediment any contaminating red blood cells. ≈ 400xg). Examples of useful density ranges for fractionating various cell populations Materials all materials required for the Percoll isolation procedure (§1.1) a continuous density gradient pouring apparatus (commercial or "home-made") Percoll solutions of the required density6 Reagents all reagents required for the Percoll isolation procedure (§1. monocytes.1 .g..step 6. washed white blood cells..p. but is is not adequate to sediment the neutrophils in the LSM. where . 6. A longer spin time (e.
The medium is best delivered from the gradient pourer into the individual tubes using a peristaltic or other pump -. 4. you will generate a gradient of continuously decreasing density. what you will notice with these gradients is that there are multiple bands. and harvest them in an analogous manner.10 METHOD 1-3. Obtain leukocyte/platelet-rich plasma as in the Percoll procedure (§1. However.as the high density medium empties from the second chamber into the centrifuge tube.1. with the density at the bottom of the gradient being equal to that of the medium in chamber 2 and that at the top of the gradient being equal to that of the medium in chamber 1. The second chamber should be equipped with a mixer or stirrer to ensure rapid and complete mixing of the reagents during this process. When the gradient formation is complete. By steadily withdrawing the delivery needle as the centrifuge tube fills. the precise number and . Pour the continuous density gradients by placing low density Percoll in the first chamber and high density Percoll in the second chamber. it is replaced and thereby diluted by low density medium. very carefully load the peripheral blood leukocytes to be fractionated on top of the gradients. great care may to be needed to avoid mixing of the cells and gradient medium. mixer peristaltic pump centrifuge tube delivery tubing or needle flow low density Percoll (chamber1) high density Percoll (chamber 2) withdraw needle with advancing gradient 5.1). 6. Since the low density medium may be of a density very similar to that of the medium in which the cells are resuspended. Run the gradients for 25 min at ≈2000 rpm in the clinical centrifuge. just as in §1.
For example. and will run as a discrete band at a density of about _____g/ml.11 7. DMEM-10% or RPMI-10%) and count them using a hemocytometer. their locations depending on the species and immune status of the blood donor. Wash the cells from each band using an appropriate medium for your purposes (e.. The cells can be morphologically identified on stained cytocentrifuge preparations. Harvest each band into a separate tube. quiescent eosinophils of humans and horses will run at a density equivalent to about _____g/ml. . while activated eosinophils are hypodense.g.
Determine the cell numbers using a hemocytometer. Using a ≈25 ga needle. through the abdominal wall. and then wet its abdomen with 70% ethanol. inject ≈10 ml of HBSS into the peritoneal cavity. 15 ml centrifuge tubes. tubes 23-25 ga needles & 10 ml syringes pipettes/pipettors (micropipettes and macropipettes) sterile surgical tools (optional. we will simply examine the method for lavage of the serosal cavity. 4. Wash the cells by centrifugation and resuspend to the desired cell density. 2. Materials BALB/c mouse anaesthetic (methoxyfluorane) clinical centrifuge.4). In this section.s. 4 ml round bottom p. in the desired medium. and then massage the distended gut to wash the serosal cells into the HBSS. 5. Euthanize mouse with methoxyfluorane and cervical dislocation. Fully reflect the abdominal skin dorsally and ventrally.12 1. for open body cardiac puncture) laminar flow hood inverted microscope hemocytometer Reagents Ca++Mg++-free-HBSS (HBSS) DMEM-10% FCS (see Appendix A) 70% ethanol cytocentrifuge METHOD 1. 3. The serosal (abdominal or peritoneal) cavity of mice is a rich source of macrophages which are easily purified by simply flushing the cavity and performing a plastic adherence step (see §1. Slowly withdraw the fluid using a ≈23 ga needle on a 10 ml syringe and transfer the wash solution to a 50 ml polypropylene tube. Repeat the wash with another 10 ml of HBSS and pool the two washings. being careful to not tear any holes in the abdominal wall (small holes discovered during step 2 can be clamped off using hemostats).2 Isolation of cells from the peritoneal cavities of mice. .
monitoring the success of washing by direct visual observation under the inverted microscope.3 Adherence purification of monocytes or macrophages Monocytes or macrophages can be easily purified by taking advantage of the fact that they adhere rapidly and tenaciously to plastic (polystyrene. To activate the cells in situ. 90 . Repeat this procedure as needed. but increases the wear and tear on the macrophage surface proteins). .2) plastic petri dishes or multi-well microscope slides DMEM-0% FCS DMEM-10% FCS METHOD 1.02% EDTA in saline. non-adherent cells by aspirating the medium (save the aspirates if you want to retain the monocyte-depleted lymphocyte preparation). To remove the cells from the plastic.wash the cells and resuspend in DMEM-10% FCS. Alternately. Macrophage adherence ostensibly works much better in the absence of protein (e.g. and then transfer the dishes onto ice for 10-15 min. the cells can be removed by treating with EDTA and trypsin (this eliminates the need for scraping. so this protocol will be performed using DMEM-0% FCS. So in this procedure. you can either remove the medium from the cells and replace it with 0.1) or peritoneal macrophages (§1. Materials/Reagents all materials for purification of mononuclear and PMN from mouse PBL (§ 1. FCS). By smoothly scraping the bottom of the dish with a cell scraper. Resuspend the mononuclear or serosal cells at ≈3x106 cells/ml in DMEM-0% 2. Remove the resuspended. 3. Remove the non-adherent cells by repeatedly pipetting the culture medium directly onto the cell monolayer that has formed on the bottom of each well. 4. If you are not thorough enough at this stage. Incubate the cells at 37˙C in the CO2 incubator for ≈3 h.95% of the adherent cells will be dislodged as viable cells .13 1. 5. large numbers of lymphocytes will remain with the monocytes/macrophages on the plastic/glass surface. FCS and add 300 µl of the cell suspension to each well of the multiwell slide. a particularly convenient format for the experiments we will be doing. just add LPS to a final concentration of ≈1-10 µg/ml. but not polypropylene) surfaces. you will simply take the total mononuclear cell fraction from PBL as well as the total peritoneal lavage population and place the cells into the wells of multi-well microscope slides..
This is the basis for antibody-dependent cellular cytotoxicity (ADCC) by immunoinflammatory cells against multiple types of cellular targets (e. Take the monocyte/macrophage monolayers.4.3).e. antibody.12) 0.[or C'-] coated) sheep red blood cell phagocytosis by purified PBL monocytes. or use peritoneal lavage cells from normal or proteose/peptone-injected mice humidified 37˙C CO2 incubator clinical centrifuge & tubes sterile eppendorf tubes Reagents C3b-coated zymosan beads (§5. are very dull by phase contrast) or remain in the extracellular compartment (i.. C3b-coated zymosan. Examine the cells using the phase contrast condenser of the inverted microscope to determine the extent to which the SRBC/zymosan particles have been internalized by the macrophages (i..or C3b-dependent phagocytosis by macrophages In this assay we will explore the potentials for macrophages to specifically interact with cells that have been targeted by the humoral immune system.. 2. Remove the slides from the incubator and wash the free SRBC or zymosan from the cultures by resuspending the sedimented cells with your pipetter and aspirating the contents. are very bright .g. normal mouse serum (heat-inactivated at 56˙C for 30 min) DMEM-10% FCS METHOD 1. in multi-well slides out of the 37˙C incubator and to pairs of the wells add either 20 µl of either: the antibody-coated SRBC.14 1. normal SRBC suspension.4 Antibody. etc.. We will use the classical example of opsinized (i.13).5% suspension of SRBC in PBS anti-SRBC-coated SRBC (§5. tumour cells.e. Incubate the cells/slides for 60 min at 37˙C.e. Materials monolayers of PBL monocytes in 8-well multi-well slides (§ 1. or 'activated' zymosan suspension.). antibody-coated cells can be phagocytosed or otherwise killed by macrophages that bind the target via the FcγR. That is... bacterial cells.4.
and stain the slides with Giemsa stain (Appendix D). and probability values) on a graph. and plot the data (including means. you will count the numbers of SRBC contained within 25 macrophages in each of ten 40x microscope fields. Allow the slides to air dry. Calculate the mean numbers (+/. To do this. Return the cells to the 37˙C incubator and periodically check them again to follow the internalization of the particles. .SEM) of SRBC or zymosan particles in the macrophages in each treatment group.15 by phase contrast). take the well casing from the slide itself and fix the cells by immersion in 100% ethanol for ≈2 min. At the end of the experiment. SEM. 3. 5. Perform an ANOVA test to determine the statistical significance of your results. for each well on the slide.
Take a look at the growing Pu5 cells under the inverted microscope to get a feeling of how they 'should' look under normal conditions. IL-6. To resuspend them in fresh DMEM-10% FCS. LPS) DMEM-10% FCS (see Appendix A) METHOD 1. dislodge the Pu5 cells from the plastic and then transfer them to a 50 ml centrifuge tube. In this protocol. a murine macrophage cell line. immune complexes. coli lipopolysaccharide. we will use the bacterial cell wall product lipopolysaccharide (LPS) to activate cultures of Pu5-1.. 2. Dispersal will . While counting the Pu5 cells. and then very briskly and repeatedly flick the tube with the cell pellet to disperse the pellet. etc. IL-1. bacterial products. precisely the same protocol and approximately the same results would be obtained if we were to use freshly purified monocytes or macrophages. Materials laminar flow hood humidified 37˙C CO2 incubator subconfluent monolayer cultures of Pu5-1.5 Activation of macrophages with bacterial lipopolysaccharide Monocytes or macrophages can be activated by the addition of many different kinds of reagents (e. 1 mg/ml DMEM-0% FCS (E.8 (Pu5) cells.8 cells in DMEM-10% FCS clinical centrifuge & tubes inverted microscope sterile eppendorf tubes cell scraper -20˙C freezer & freezer bags Reagents bacterial endotoxin.g.e. While we will be using this cell line (in order to save the lives of a number of mice).). Later in the week we will then determine the extent to which you have activated the cells by quantifying their secretion of a number of monokines (i. and TNFα).16 1. interferon-γ.. sediment them by centrifugation (10 min at ≈1500 rpm in the clinical centrifuge). Using a cell scraper. completely aspirate the supernatant from the tubes. Pu5 cells were one of the original lines used in the cloning of murine TNFα. 3. Remove 20 µl of the cell suspension for cell counting with the hemocytometer..
and then dispense the cell suspension into the wells of 24-well plates. place all of the plasticware & cells in the contaminated-discard pan. . centrifuge them for a few minutes at full speed in the microfuge. 6. Pu5 supn't. the date. Label the eppendorf tubes with your initials. Return the cultures to the 37˙C CO2 incubator. as well as 4 wells which contain medium but no cells (LPS-medium controls) Add LPS (to a final concentration of 10 µg/ml) to the 4 'stimulated cells' wells and to the 4 'LPS-medium control' wells.. save several aliquots of the DMEM-10% FCS that was used for the cultures (as medium controls) At 1. at 1 ml per well. In addition to the supernatants from the cell cultures. 7. At each time. Store all aliquots in the -20 freezer (long-term storage requires a 80 freezer). and 24 h post-challenge. examine the cells to get a feeling for whether the LPS has affected them in any visible manner.g. 6. Add sufficient DMEM-10% FCS to bring the cells to a concentration of 3x106 cells/ml. 1 [or 6. + [or -] LPS. be complete when the pellet has become a paste on the walls of the tube.17 4. or 24] h). also harvest the culture medium from the appropriate wells. At the end of the experiment. and an equivalent amount of DMEM to the 4 wells of unstimulated Pu5 cells. 5. You will need 8 wells of cells (4 wells of unstimulated cells & 4 wells of stimulated cells) for todays experiment. and then aliquot each one into four eppendorf tubes (200 µl/tube). and the sample information (e.
Before the assay.G4) to proliferate in the presence of IL-1. proliferate in response to IL-1 in the presence of sub-mitogenic doses of PHA or ConA.5-diphenyltetrazolium bromide). not the numbers of cells. and which were washed and held overnight in Click's-10% FCS without conditioned medium (i. While this is a very convenient parameter. we will depend on the property of LM-1 cells (a sub-clone of the ATCC cell line D10.6 Monokine Bioassays: 1. such as the influence agents that affect mitochondrial activity can have on the results.e.1 Assay for IL-1 activity In this assay. tips multi-channel pipetter clinical centrifuge.6. The cell proliferation will be measured by examining the abilities of the cells in each well of the 96-well plates to take up and reduce the dye MTT to an insoluble blue-black formazan precipitate within their mitochondria. While D10.G4 cells. tubes 15 ml 15 ml centrifuge tubes hemocytometer ELISA plate reader (with a 595 nm wavelength filter) Reagents LM-1 cells which have not been fed fresh IL-1-containing medium for 5-7 days.18 1.5 ng/µl) MTT. Materials humidified CO2 incubator 96-well tissue culture plates micropipetters. Thus the assays measures the mitochondrial activity of the cells. (3-[4. LM-1 cells do not need the PHA or ConA to respond to IL-1. like thymocytes. IL-1-starved LM-1 cells) Click's-10% FCS without conditioned medium (Click's-10% FCS-CM-) recombinant mouse IL-1 (our present stock soln is at a concentration of 7. it carries with it some problems.. 5 mg/ml in PBS (stable for 2-3 wks at 4˙C) acidified isopropanol (see Appendix C) . the cells are rendered more sensitive to IL-1 by starving them of this cytokine for ≈5-7 days.5-dimethylthiazol-2-yl]-2.
. but which has not been exposed to the experimental cells). -In this format. H1) of each plate. . using a template pattern similar to the one depicted. -Always include a medium-only control (i.5.0. Starve the LM-1 cells 4-7 days before assay (feed them fresh conditioned medium some 4-7 days before the assay. each done in quadruplicate.19 METHOD 1. but do add all other reagents (e.and dispense into the 96-well plates at 100 µl/well.if you have multiple doses of test samples. wells A1...e.. in quadruplicate sets of wells) to a series of wells to achieve final doses of 0. wash the cells in fresh Click's-10% FCS-CM.g. On the day of the assay. and then do not feed them again). one plate will accept 10 experimental samples. but add DMEM-0% FCS to these wells.5 5. B1. Add 80 µl of Click's-10% FCS-CM-. 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 standards (pg/ml) 0. Click's without IL-1) -. 2. 50 and 500 pg/ml (see Appendix D.0 50 500 plate blank (A1-H1) cytokine standards (4 doses) test samples (10/plate) medium control 'plate-effect' blank wells 3. Use the same volume of experimental medium as that used in the test samples wells -. Generation of standard curves)..and leave them in culture overnight. The night before the assay. resuspend the LM-1 cells to 4X105 cells/ml in Click's10% FCS-CM. use equivalent multiple doses of medium controls. -Do not use the outer wells of the plate for any samples (there is an 'edge-effect' of the plates). 5.this will be the plate blank. the same medium as that used for the experimental treatments. . -Do not add any cells to the first column (i..e. and 20 µl of appropriately diluted rmIL-1 standard (like all samples and standards.
and then return the plates to the humidified 37˙C CO2 incubator for 1 . and then read the plates on the ELISA plate reader. Plot your results as a bar graph (+/.20 4. 6. To measure the extent of the IL-1-driven LM-1 cell proliferation in each well. set at a reading wavelength of 595 nm. Download the data to a 3-1/2" Macintoshformatted floppy disc. . and Statview+ to perform the statistical analyses. Use the Microplate manager program to crunch your data. 5. Bring the final volume of each well to 200 µl with Click's-10% FCS-CM-.SEM) using the Cricket III program provided. (Optional: centrifuge the plates for 10 min at 1500 rpm to sediment cells. run samples (LPS-stimulated Pu5 cell culture supernatants) and medium controls (DMEM-10% FCS + 10 µg/ml LPS) at required volumes (usually 5-20 µl/well). Agitate the plates on the ELISA plate shaker for ≈3 minutes. In parallel sets of quadruplicate wells. and add 100 µl of acidified isopropanol to each well.) Carefully remove 150 µl of medium from each well in the plate.5 days. and return the cells to the 37˙C humidified CO2 incubator for 3 . use the micropipetter to add 20 µl of MTT stock solution (5 mg/ml) to each well.2 h. 7.
0 or 5. so you will need three sets of 'medium controls'. and resuspend to a concentration of 2. 250 & 2500 pg/ml final concentration) in 20 µl of RPMI-10% FCS. 2.5.2 Assay for IL-6 activity Like the IL-1 assay.6. we will use 1. 2.21 1. and return plates to the incubator for ≈1-2 hr.0 µl of the LPSstimulated Pu5 cell culture supernatants. Return the plates to the 37˙C CO2 incubator for 3 days. In this assay. Again.0. 3. Add 100 µl of cells to each well in plate. the IL-6 assay depends on the fact that 7TD1 cells proliferate strongly in response to low concentrations of IL-6. and 5. After 3 days.0.0 µl of DMEM-10% FCS supplemented with 10 µg/ml of LPS. Materials humidified CO2 incubator 96-well tissue culture plates micropipetters.1 (IL-1 assay). using the same or a similar sample plate format to that indicated in §1.0. When the . Wash the 7TD1 cells two times in RPMI-10% FCS. and add your samples and experimental medium controls in appropriate volumes. we will measure this response using the MTT dye method. each containing 1. 5. our stock solution is at 100 ng/ml) MTT (5 mg/ml in PBS) acidified isopropanol METHOD 1. 4. tubes 15 ml 15 ml centrifuge tubes hemocytometer ELISA plate reader (with a 595 nm wavelength filter) Reagents 7TD1 cells in RPMI-10% FCS supplemented with rhIL-6 (80 pg/ml) RPMI-10% FCS (see Appendix B) recombinant human IL-6 (rhIL-6.5x104 cells/ml in RPMI-10% FCS. Add cytokine standards (2.5. add 20 ul of MTT solution (5 mg/ml) to each well (including plate blank wells). tips multi-channel pipetter clinical centrifuge. 2. 25.
mitochondria in each of the cells are plainly visible at 40x magnification.SEM) using the Cricket III program provided. and Statview to perform the statistical analyses. 7. read the plates at 595 nm wavelength on the ELISA plate reader and download the data to a 3-1/2" Macintosh-formatted floppy disc. and then leave the plates on your bench overnight. The next morning. Use the Microplate manager program to crunch your data. vortex vigorously in the ELISA plate shaker for ≈3 min. . Add 100 µl of acidified isopropanol (lysis buffer) to each well.22 6. centrifuge the plates and remove 150 µl of medium from each well. Plot your results as a bar graph (+/.
when they are beginning to lift well. . tips multi-channel pipetter clinical centrifuge..g. but many still remain attached. Materials humidified CO2 incubator 96-well tissue culture plates micropipetters. FCS). All cells will be dislodged instantaneously. Add ≈4 .23 1. DMEM-0% NHS recombinant murine TNFα standards (diluted as in Appendix D) Actinomycin-D (stock 5 mg/ml in 95% ethanol) MTT (5 mg/ml PBS) Acidified isopropanol (lysis buffer) METHOD 1. the assay should be run the absence of other proteins. Finally.5 drops of 1% trypsin/≈10 ml of medium and allow the typsin to digest the cells off of the plastic.3 Cytotoxicity assay for TNFα bioactivity TNFα activity is usually detected using a cytotoxicity assay. tubes 15 ml 15 ml centrifuge tubes hemocytometer ELISA plate reader (with a 595 nm wavelength filter) Reagents L-929 cells in DMEM-10% normal horse serum (NHS). the cytotoxic effects of this cytokine are markedly diminished by the presence of other proteins (e. forcefully slapping the flask down on your thigh. so that as much as possible. actinomycin D)..g. On the day before the assay. harvest L929 cells from a flask by trypsinization (remove DMEM-10 % NHS medium and add DMEM-0% NHS medium. L929 cells are most sensitive to the effects of TNFα when the assay is run in the presence of a low levels of a transcription inhibitor (e. L929 cells (or at least many of its sub-lines) are sensitive to TNFα such that this cytokine kills the cells over ≈18 h. This process can be expedited by watching the cells and. However.6.
8.5 µg/ml of actinomycin D (i. Return plate to the 37˙C CO2 incubator.1. Add 20 µl of MTT (5 mg/ml PBS) to each well (including plate blank wells). control medium.04. Perform a statistical analysis to confirm that your results are meaningful. examine the cells under the inverted microscope to get a feeling for the relative levels of L929 cell death in each well. After 45 . so that the total well volumes equal 200 µl. 6. 7. Calculate the cytotoxicity in each of the wells using the formula: percent cytotoxicity = mean OD590 medium control wells . and experimental samples (LPS-stimulated Pu5 cell supernatants & controls) to their appropriate wells. express them in terms of units (+/.5. 4. 4. remove all of the serum-containing medium from the wells by inverting the plate and vigorously flicking it. re-examine the cells to confirm that adequate levels of MTT conversion to formazan dye have occurred in the mitochondria (see §1. just prior to running the assay. The next day.5. IL-1 assay) and then remove 150 µl of medium from each well in the plate and replace it with 100 µl of acidified isopropanol. Return plate to the 37˙C CO2 incubator overnight. Wash the dislodged cells in DMEM-10% NHS medium.60'.24 2. Vortex the plates on an ELISA plate shaker to solubilize the formazan dye. Add TNFα standards (0. each in a 20 µl total volume.SEM) of TNF activity (a unit of TNF activity is that amount of cytokine required to kill 50% of the cells in the assay) and graph your results.e.0 and 40 units/well). a 2000-fold dilution of the stock Act D). 5. resuspend them to 4..SEM) cytotoxicities for the standards and each treatment group. IL-1 assay). The next day. and again return the plates to the incubator.1.OD590 experimental well x 100 mean OD590 medium control wells 3. 9. 0. Calculate the mean (+/. and read the plates on the ELISA plate reader at 595 nm wavelength. see §1.5x105 cells/ml. and dispense 70 µl to each well of a 96-well plate (for plate format.4. Add 180 µl of DMEM-0% NHS containing 2. .
11 ). Prepare the chemoattactants for use in the assay...g. IL-8 or CINC in rodents).4. where they are important to the clearance of bacteria or other insults both by virtue of their abilities to phagocytose the offenders.11) interleukin 8 (or CINC) METHOD 1.§5.25 1.g.g. as represented in zymosan-activated sera (i. and an array of chemokines (e.g. sedimentation (or if your experiments require purified cells. For this exercise.4.1. the fMLP to final . as well as through their toxic secretory products. Generate a neutrophil-rich population from the peripheral blood. loci of C' cascade activation. 1:1000.e. Neutrophil chemotaxis assay Neutrophils are attracted in large numbers into inflammatory foci (e. allowing approximately 30 µl of each chemoattractant dilution. zymosan-activated serum. C3a. tips purified peripheral blood granulocytes Reagents DMEM-0% FCS media Ca/Mg--HBSS f-met-leu-phe bacterial tripeptide zymosan-activated serum (§ 5. using dextran 2. adjusting the population to a final concentration of 2x106 cells/ml of HBSS.. Millipore or NeuroProbe) micropipetters. C5a).7. Materials humidified CO2 incubator microchemotaxis chamber (NeuroProbe Inc) PVP-free (PVPF) polycarbonate cell migration filters (5 µm pore size.. C3a. Dilute the zymosan-activated serum (ZAS) to final concentrations of 1:10. including bacterial products (e. C5a). bacterial infections). fMLP).. complement split products (e. 1:100.e. percoll-purified neutrophils).. then use these cells to study the chemoattractant activities of fMLP and C3a/C5a (i. formyl-methionyl-leucyl-phenylalanine. we will purify neutrophils from the peripheral blood of mice as in §1.. These cells have receptors for an array of chemoattractants. and 1:10000.
as first estimates with unknowns. . and the recombinant IL-8 to concentrations ranging from 50 pg/ml to 1000 ng/ml. if not quadruplicate) add sufficient chemoattractant to completely fill the wells (do not overfill them. 1:80 and 1:160.) Place the plastic gasket on top of the membrane. Put the tip of your pipette just at the surface of the Nucleopore membrane. this will become easy for you..5-4 hr incubation times to respond. 4. concentrations of 10-6. allowing them to drop off the membranes into the bottom chambers after completely traversing the porous membranes . 7. disassemble the apparatus and carefully clamp the ends of the membrane with "bull-dog" or other suitable clamps. and the top half of the apparatus on top of the gasket and firmly screw the lug-nuts down. Place the PVPF-free Nucleopore membrane (shiny side up) on top of the wells.26 3. then remove the cells which settled onto the upper surface of the membranes in each well by scraping the upper surface across a scraper (e. (PVPcontaining membranes will not retain cells that migrate completely through the pores in the assay. Add 50 µl of the responder cell population to each well. without contacting it.e.with a bit of practice.thus. a complete assessment of the chemotactic response with these membranes would require enumerating the cells associated with the membranes. such the a tight seal is created in each well. Place the chambers in a plastic dish containing damp paper towels in the 37˚C CO2 incubator.a very slight convex surface to the sample is ideal).g. To the bottom chamber of each well (samples should be run in duplicate. Upon completion of the incubation period. and 10-10 M. Eosinophils also respond in this time frame (i. 6. ≈90 min) while monocytes and lymphocytes will call for 2. 1:20. 10-8.. 5. 10-7. being careful not to smear the chemoattractants between wells. a fairly sharp-edged glass microscope slide clamped into a ring-stand clamp). being careful to not generate air bubbles which will create an air-lock on top of the membranes (and thereby exclude cells from the membranes). allowing 90 min for the neutrophils to respond to the chemoattractants. Putative chemoattractant-containing biological samples should be diluted to 1:10. as interwell smearing of chemoattractant may occur when placing the polycarbonate membrane is step 4 -. 8. 10-9. 1:40. and quickly expel the 50 µl of chemoattractant . as well as those in the lower chambers.
then stain with a suitable staining solution (e. so that the cells which have migrated completely through the membranes are most apparent.. Count the cells with have migrated completely through the membranes. The nuclei of the cells that are still within the membrane pores will be visible as dark blue or purple shapes within the otherwise clear-tolight purple pores. as well as those within the pores . 10. Diff-Quick).27 9. Mount the membranes with the bottom side of the membranes uppermost. the membranes can be dipped in xylene to "clear" them and mounted in "Permount" or other mounting medium on glass slides. Allow the membrane to air-dry. After air-drying again.g.
and higher levels of TNF than LPS-stimulated macrophages. skin. Materials humidified CO2 incubator T75 flasks hemocytometer micropipetters. For this class. tips clinical centrifuge.7 The 2 hour supernatants from these cells will contain abundant TNFα.. Mast cells can be activated by cross-linking (i.g. Cl. DNP30-40HSA(stock solution.MC/C57. and the cells will contain very high levels of TNFα mRNA. but also perhaps some less obvious ones.e. use at 50 to 100 ng/ml). FcεRI-dependent activation of mast cells Mast cells are found in increased numbers at the host's interface with its environment (e. METHOD 7 Expression of the FcεRI is inducible in mast cells.1 cells). bridging) adjacent FcεRbound IgE molecules on the cell surface. so in these experiments we will sensitize some Cl.1 cells produce higher levels of IL-4 than purified. tubes 15 ml polypropylene 4 ml culture tubes Reagents DMEM-10% FCS media Cl. Thus. fully differentiated Th2 lymphocytes. MAb IgE anti-DNP (ascites fluid. 1 mg/ml.. airways.8.MC/C57. and this is at least in part regulated the concentrations of exogenous IgE.g. allergen-reactivity). and then challenge with DNP-conjugated human serum albumin (DNP30-40HSA). can be best sensitized with IgE of the desired specificity by incubating the cells overnight in high concentrations of the antibody. such that in the presence of high concentrations of IgE.1 cells with a monoclonal IgE anti-DNP antibody. which will likely have the vast majority of their existing FcεRI occupied by IgE antibodies of irrelevant specificities.28 1.MC/C57. mast cells express more IgE receptors.1 cells in DMEM-10% FCS. use at 1:3000 for Cl. .MC/C57.. intestinal tract) and seem to subserve a number of obvious functions (e. freshly purified tissue mast cells. we will take advantage of the fact that they produce very high levels of some cytokines and use them as a positive control for some of our experiments.
cap the tubes tightly and lay them on their side in the incubator (or use a slowly moving rotator at 37˙C).1 cells and adjust the cell concentration to 3 X 106 cells/ml. 0.1 experiment). At each of the indicated times after allergen challenge (i.5. and incubate the cells for 30 .MC/C57. Wash the cells two times with DMEM-10% FCS and resuspend them again at 3x106 cells/ml. 2.29 1. exchange CO2 into the tube).5 ml of mast cell supernatant/time point. The cells can be either discarded (as in our first Cl.MC/C57. and 4 h).e. Bring the cells to 37˙C by placing them in a water bath. 9x106 cells in 3 ml).e. Freeze the aliquots at -20C (or -80C for longer term storage).20 min to allow them to gas (i.. Add DNP30-40HSA to the cells to a final concentration of 10 ng/ml and place the tubes upright and lightly capped in the 37˙C CO2 incubator for 15 . 2.. 4. Add MAb IgE anti-DNP to the tube of C57 cells to a final IgE dilution of 1:3000. our Northern analysis experiments).60' at room temperature in order to saturate the cells' high affinity IgE receptors. 1. remove a tube of cells from the CO2 incubator and sediment the cells by centrifugation (≈8-10' @ 1500 rpm).g. and then aliquot the supernatants into labelled eppendorf tubes (100 µl/tube). .. or processed for total cellular RNA extraction (e.e.. After this. You will only need ≈0. Obtain some Cl. for our in situ hybridization experiments). fixed (e. 3..g. so set up ≈3 ml of cells (i.
1 Hybridoma cell culture with production of monoclonal antibodies Materials T75 tissue culture flasks clinical centrifuge and 50 ml polypropylene tubes Reagents GK1. we will grow up anti-mouse CD4 and antimouse CD8 monoclonal antibody-producing hybridoma cell lines.5 cells. 2.000 rpm in the RC-5B superspeed centrifuge to sediment all particulate matter. 2. and analyse the purified antibodies and total culture supernatants by polyacrylamide gel electrophoresis and Western blotting. 4. one each for the TIB 211 and GK1. 75 cm2) tissue culture flasks. Aliquot the supernatants and either process expeditiously or store at -20˙C or 80˙C (for longer-term storage). purify the IgG antibodies from the anti-CD4 cell culture supernatants by affinity chromatography. Set up two T75 (i. terminal cultures). at a cell density of ≈105 cells/ml of RPMI-5% FCS medium.30 2. We will follow the success of depleting each of these populations using two-colour fluorescence activated cell sorter (FACS) analysis of the spleen cells. .7 days.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: In the second week of the course. We will then functionally characterize the antibodies by using them for negative selection of CD4 and CD8 cells from mouse splenocyte populations. Allow the cells to continue growing until the medium has gone completely yellow (acidic) and the cells have essentially all died (about 5 . Collect the supernatant from each flask and centrifuge it for 15 min at 12. Start each flask as a ≈40 ml culture..e.5 (anti-CD4) and TIB 211 (anti-CD8) hybridoma cells RPMI-10% FCS METHOD 1. 3.
or 5-10 ml syringe and glass wool dialysis tubing centrifugal concentrators (e. thereby binding the IgG antibodies to the matrix. centrifuge it for 15 min at 2.2. we will pour minicolumns of the Avid-AL matrix and run the hybridoma supernatants over the columns.1) and 2. Thus.500 rpm in clinical centrifuge to sediment all particulate matter.5 cells (see §2. dialyse it against PBS and concentrate it if necessary. Briefly. we will use it to purify the rat anti-mouse CD4 IgG antibodies from the GK1. Centricon tubes) Reagents AVID-CHROM Ig pure kit for purification of IgG -column binding buffer (>1M proprietary salt) -neutral elution buffer (1M Tris [pH 7. Shake the tube vigorously to disperse the matrix and then sediment the matrix by . Collect the supernatant from a 4 day culture of GK1.2 Affinity purification of IgG antibodies 2. Materials T75 tissue culture flasks clinical centrifuge and 50 ml polypropylene tubes AVID-AL IgG affinity column matrix 10 ml polyprep column. we will elute the IgG using a low pH buffer.5 ml of a 50% matrix slurry into a 15 ml centrifuge tube and add ≈10 ml of regeneration buffer (methanol). neutralize the eluate.31 2. Set up the affinity matrix column by pipetting ≈1.4.2 .7. and then filter the supernatant through a 0.45 µm filter. pH the supernatant to 7. including rat.5 hybridoma supernatants.4].g. Following a washing step to remove the non-specifically bound proteins.5 (anti-CD4 hybridoma) cells in RPMI-10% FCS PBS METHOD 1.. 20% glycerol) -regeneration buffer (buffered methanol) GK1.1 Avid-AL affinity chromatography AVID-AL is a popular new matrix with a natural affinity for IgG from a wide array of species.
determine the protein concentration of the eluted IgG solution using a Coomassie Brilliant Blue (AKA Bio-Rad or Bradford) protein assay (Appendix D) and. salts will precipitate with long term storage) or PBS (for longer term storage). Wash the column with ≈10 ml of binding buffer. collecting the eluate into one tube. Elute the IgG from the column by running 4 .). 4. Resuspend the matrix in ≈5 ml of binding buffer and pour this into a polyprep column. centrifugation. or until the phenol red from the culture supernatant fluid has leached from the matrix (it will turn from a brown to the lime-green colour). Dialyse the elute IgG overnight against three changes of PBS (≈1 liter ea.5 ml of the neutral elution buffer (1 M Tris [pH 7. After dialysis. 6. 5. concentrate the eluted protein using a centrifugal concentrator. 20% glycerol) through the column.32 3. Regenerate the column with ≈10 ml of regeneration buffer. and store in either binding salt (very short term storage. Dilute the hybridoma culture supernatant fluid with 2 volumes of binding buffer (to bring the final salt concentration to ≈500 mM).5].20 ml of binding buffer. and then run the supernatant through the affinity column matrix several times to saturate the IgG binding capacity of the matrix. Wash the matrix with ≈15 . if necessary. .
Pipette ≈1 ml of the protein A-Sepharose 50% matrix slurry into the column and wash it with several column volumes of PBS. using a spectrophotometer or UV monitor (OD 260) to confirm the end-point.0. of course) as affinity matrices for this purpose. 2.2 . and IgG3 at pH 3. or Sigma) clinical centrifuge and 50 ml polypropylene tubes 10 ml polyprep column. or 5-10 ml syringe and glass wool spectrophotometer or UV monitor (equipped for reading OD 260) dialysis tubing Reagents 0.5.5 culture supernatant through the column matrix several times to saturate the IgG binding capacity of the protein A. The elution pH optima of IgG from the protein A columns is isotype-specific. IgG1 antibodies will only bind to protein A at pH 8.2 Protein A-Sepharose affinity chromatography Protein A from Staphylococcus aureus (Cowan strain) is a molecule with a very high affinity for IgG antibodies. Filter the GK1.4.5 (rat IgG2a anti-CD4 hybridoma) cells PBS (pH 7. In general. Run the GK1.0) METHOD 1. while IgG2a and other IgGs will do so a more physiological pH (i. 4.45 µm filter and pH it to 7.5) supernatant from a terminal culture of GK1. pH 7. 3.2.0. and has been used for several decades as the protein of choice to purify these antibodies.5 culture supernatant through a 0.1 M citric acid (pH 4.e. Wash the column with PBS until no more protein elutes from the column. We will use recombinant protein A bound to dextran beads (Sephadex-G50).2) as well as pH 8.2) PBS/20% ethanol 1M Tris (pH 9. with IgG1 elution being best performed at pH 6.0 (the least hostile or acidic elution conditions available should be employed to prevent acidic hydrolysis of the antibodies) Materials protein A-Sepharose (Pharmacia. IgG2a at pH 4. .5.33 2. but in the past people have used the bacteria themselves (fixed..7.
Elute the IgG from the column by running ≈10 ml of the 0.1 M citric acid elution buffer through the column, collecting the eluate into one milliliter fractions (i.e., 1 ml/tube). In order to minimize the time that the antibodies remain in an acidic environment, we will elute the column directly into 1 M Tris buffer (pH 9.0). Determine the protein contents of the eluted fractions by either determining the OD260 of the fractions, or by use of a protein assay (e.g., Coomassie Brilliant Blue [a.k.a. Bio-Rad, Bradford or CBB] protein assay; Appendix D), and pool the protein-containing fractions. Regenerate the column with ≈10 ml of PBS, and store the matrix in PBS/20% ethanol. Dialyse the eluted IgG overnight against three changes of PBS (≈1 liter ea.). After dialysis, determine the protein concentration of the eluted IgG solution using a and, if necessary, concentrate the eluted protein using a centrifugal concentrator.
2.3 Preparation of IgM antibodies IgM antibodies do not adhere well to most of the available immunoglobulin
affinity matrices (e.g., protein A, T gel, Avid-Chrom), so alternate methods must be employed to prepare IgM antibodies. A number of methods are available, with the simplest true purification probably being achieved by size exclusion chromatography (IgM pentamers have a molecular mass of ≈750 kD, while IgG and albumin have molecular masses of 150 & 65 kD, respectively). In this session, we will simply enrich for IgM antibodies by differentially "salting out" the IgM protein with ammonium sulfate. High concentrations of ammonium sulfate will cause the proteins in a solution to differentially precipitate out from their solubilized state - at 30% ammonium sulfate saturation, most of the IgM antibodies will precipitate out of solution, while at 45% saturation most of the IgG isotypes and the residual IgM antibodies will precipitate out of solution.
Materials beaker and stir bar
clinical centrifuge & tubes dialysis tubing 0.45 µm filters pH meter and pH reagents stir plate syringe and 20 ga needle
Reagents culture medium from a terminal culture of TIB211 (IgM anti-CD8) hybridoma cells saturated ammonium sulfate solution borate-buffered saline Method 1. As with the IgG purification, generate a 4-day TIB211 hybridoma culture
supernatant, sediment the particulate matter by centrifugation, then filter and pH the supernatants as in §2.2. Place the supernatant in a beaker on a stir plate and drip saturated ammonium sulfate into the supernatant, while constantly stirring, to a final ammonium sulfate concentration of 30% (i.e., 0.5 volumes of ammonium sulfate into 1
volume of antibody). Use a syringe and 20-23 ga. needle (as required) to drip the ammonium sulfate into the antibody culture supernatant, drop-by-drop. After the 30% ammonium sulfate has precipitated the available proteins in the hybridoma supernatants, allow the stirring to continue for an additional 30 min, then sediment the precipitate by centrifugation (15 min at 2500 rpm). Return the supernatant to the beaker and continue dripping ammonium sulfate into the supernatant to a final ammonium sulfate concentration of 45%, and then sediment that as in step 3. Dissolve the precipitated proteins in borate-buffered saline, dialyse overnight versus an excess of borate-buffered saline, determine the protein concentration using a CBB assay and aliquot and freeze.
We will use polyacrylamide gel electrophoresis (PAGE) for the former. and 7. and assemble the PAGE apparatus.4 Analysis & characterization of immunoglobulins In this section. as well as the TIB211 anti-CD8 IgM antibodies and characterize them according to their size (molecular mass) and reactivity with anti-IgG and -IgM antibodies. Mix together in a side-arm flask. Add 150 µl of ammonium persulfate.1 Polyacrylamide gel electrophoresis of immunoglobulins PAGE is a powerful technique for confirming the presence of proteins in solutions. 2.9 ml of the acrylamide/bisacrylamide solution. 10% acetic acid) rapid Coomassie blue stain (0.8% bisacryl) Commercial separating and stacking gel buffers ammonium persulfate 10% solution (freshly prepared) isobutyl alcohol (H2O-saturated) PAGE 2x sample prep buffer (for denaturing & reducing the samples) PAGE 5x SDS/run buffer (dilute 1:5 with H2O before use) PAGE gel fix solution (25% isopropanol. and mix once again by swirling gently. although it is lacks the specificity of Western blots in identifying the proteins (beyond their molecular weights). 3. Degas the solution under vacuum for 10 . Clean the glass plates and gaskets. However.006% Coomassie Brilliant Blue G-250 in 10% glacial acetic acid) protein samples biotinylated molecular weight markers (for Western blots) unstained molecular weight markers (for protein staining gels) METHOD 1. 2.4. we will take the anti-CD4 IgG that you have purified. and Western blotting for the latter.75 ml of the separating gel buffer.15 min.37 2. 3. and pour the acrylamide separating gel mixture . Place a mark on the glass at the 6 cm mark (from the bottom). Materials PAGE mini-gel apparatus and power pack Reagents Commercial acrylamide/bisacrylamide solution (40% acryl/0.2 ml of H2O. it is an ideal method to confirm the homogeneity or lack thereof of purified proteins.
For complex mixtures of proteins. so that loading 5 µl of marker mix will give you 5 µg of each band in the mixture. 12 The wells of the gels will be able to hold ≈30 µl of sample/sample prep buffer . Remember to run the appropriate molecular weight markers10. Place this assembly into the PAGE run reservoir and fill the reservoir and top of the gel assembly with PAGE run buffer. a ≈1:20 final dilution of the mix). 8 For non-reducing conditions. and then pour off the alcohol overlay and rinse the top of the gel with water. Turn off the power. while for ammonium sulfate precipitated IgM-containing culture supernatants. 10 Unstained molecular weight markers from Gibco/BRL will contain ≈1µg of each marker protein per µl of solution. the buffer should contain ≤10% 2-mercaptoethanol.5 ml of the acrylamide/bisacrylamide solution. the sample prep buffer does not contain 2-mercaptoethanol. Carefully pipette each sample into a well of the gel. you would need to run perhaps 20 µg of protein.e. 4. To do so.15 min in isopropanol fix solution. Thus. Attach the gels/plates to the electrode frame and seal the seams with agarose. 7. while for reducing condtions. 8.15 min. and then overlay the mixture with H2O-saturated isobutyl alcohol. Incubate the gel for 10 . mix the protein sample 1:1 with sample prep buffer8 in an eppendorf tube and place in a boiling water bath for 5 min9. disassemble the PAGE gel apparatus.12 Run the gel at 150 volts until the bromophenol blue dye front reaches the bottom of the gel. with the teeth submersed in the stacking gel solution. the gel can be dried down using a commercially available drying apparatus. stain it in the rapid Coomassie brilliant blue for 2 h to overnight at room temp. and pipette this solution on top of the polymerized separation gel. 11. and degas the solution under vacuum for 10 .38 into the PAGE apparatus up to the 6 cm mark. In another side-arm flask. 3. Immediately place the well-forming comb in place. Pulse microfuge the tubes to sediment samples and hold on ice until running them on the gels. you could run only 1-2 µg. for protein A-purified IgG. you will need to run greater amounts of protein in order to visualize the multiple bands in the samples. mix together 0. load 1. 11 For Western blots which are to be probed with an avidin-alkaline phosphatase detection system. Allow the PAGE gel reagents to polymerize.5 ml of the stacking gel buffer.. 9 Load the proteins such that individual protein bands should contain ≈1-2 µg of protein. Prepare samples for the PAGE run while the stacking gel is polymerizing. Add 25 µl of ammonium persulfate. 6. 4. and then destain in several changes of 10% glacial acetic acid over 5-8 h. Allow the stacking gel to polymerize. and then remove the comb from the top of the stacking gel. 5. and mix once again by swirling gently. For permanent storage.5 µl of marker mix/lane (i.
Western blotting is a very sensitive method of detecting proteins to which antibodies already exist.4.1% Tween 20) TTBS-5% Carnation skim milk powder PBST (PBS with 0. with two 'brillo' pads flanking the filter paper.or horse radish peroxidase-labelled antibodies and appropriate chromogens. This arrangement of gel. nitrocellulose membrane and pads (depicted below) is clamped between the Western blotting electrodes such that the gels is on the negative electrode side and the nitrocellulose is on the . 2. Materials Western blotting transfer apparatus (wet) Whatman #1 filter paper nitrocellulose transfer membrane (do not handle with bare hands) Reagents PAGE gel with separated proteins to be transferred (unfixed) Western blotting transfer buffer TTBS (Tris-buffered saline with 0. proteins that have been fractionated on PAGE gels are electrophoretically transferred to nitrocellulose or other types of membranes (e. Equilibrate the gel after electrophoresis to Western blot transfer buffer by incubation for ≈45 min. derivatized nylon). diluted 1:5000 in PBST) METHOD 1.39 2..g. Also equilibrate a sheet of nitrocellulose (cut to the same size as the half the gel to be used for Western blotting) to the same buffer.2 Western blotting to detect immunoglobulins In Western blotting. such that the gel is sandwiched immediately next to the nitrocellulose (with no air bubbles between the two) and both are sandwiched between two sheets of filter paper. to which they may differentially bind.05% Tween 20) bromo-chloryl-indoyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate streptavidin-alkaline phosphatase conjugate (strep-AP. using alkaline phosphatase. Transfer the gel to the gel blotting bracket. and then the protein bands on the membranes are visualized immunochemically.
5). 9. Disassemble the transfer assembly and stain the gel as in §2. add 22 μl BCIP. fill it with transfer buffer and then move the whole apparatus to the cold room. and then wash the blot with H2O and air dry.05 M MgCl2. Transfer the nitrocellulose membrane into a large weigh boat containing 15 ml of TTBS-5% Carnation skim milk powder for 2 h to overnight to block the non-specific binding of other proteins to the nitrocellulose membrane. Wash the blot three times 15 min in H2O.40 positive electrode side (the proteins will be negatively charged due to the SDS in the PAGE sample prep buffer). 'brillo' pads negative electrode Whatman #1 filter paper PAGE gel nitrocellulose ASSEMBLED TRANSFER ASSEMBY positive electrode - + 3. Incubate for 30 . 5. 7. Set the gel assembly in the gel apparatus. and use the plot and the migration distances of the bands in the IgG and IgM lanes to interpolate the relative molecular weights of the proteins detected. Plot the relative migration distances of each of the molecular weight marker proteins on a graph. Run the transfer at 100 volts for 23 h (IgM may migrate through the nitrocellulose membrane in 3 h). and thereby confirm their identities. 4. (To prepare the BCIP-NBT substrate: to 5 ml of 0.1 M Tris (pH 9. 0. mix by inverting then add 16. 0. 6.5 μl NBT and again mix).1. Wash the blot three times 5 min in PBST and place the blot in 15 ml of PBST containing a 1:5000 final dilution of streptavidin-alkaline phosphatase conjugate for 90 min at room temperature. and then transfer into 15 ml of freshlyprepared TMS -BCIP/NBT mixture. Wash the membrane two times for 5 min each with PBST and place the blot into 15 ml PBST containing biotinylated rabbit anti-rat IgG and IgM (1:1500 final dilution) for 60 min at room temperature.40 min until the bands become well-defined. 8. .1 M NaCl.4.
15 and 50 ml polypropylene tubes Reagents GK1. medium only 100 100 100 100 100 .1 COMPLEMENT (µl) 0. respectively. Materials single cell suspension of mouse splenocytes (at 1x107 cells/ml. we will learn how to deplete selected populations of cells from a heterogeneous mixture of cell types.5 (anti-CD4 hybridoma) cell supernatants (from a 2-4 day culture) TIB211 (anti-CD8 hybridoma) cell supernatants (from a 2-4 day culture) purified GK1.0 T CELL AND B CELL RESPONSES: 3. Label and set up a series of tubes to receive reagents as follows: LABEL no Ab. no C' HB 121 (control) GK1. specificity: mouse IgG1 anti-human IgE) fluorescein-labelled anti-mouse CD4 IgG antibodies phycoerythrin-labelled anti-mouse CD8 IgG antibodies Low-tox rabbit C' (Cedar Lane) METHOD 1.0 0.41 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells In this exercise. We will confirm this depletion by using commercially available fluorescein-labelled anti-CD4 and phycoerythrin-labelled anti-CD8 antibodies to stain the residual cell populations and then we will analyse the composite phenotype of these cells using two-colour FACS (fluorescence-activated cell sorter) techniques.5 supn't GK1.5 IgG antibodies purified HB121 IgG antibodies (control IgG. Specifically.5 IgG CELLS 100 100 100 100 100 100 ANTIBODY (µg/ml) 0 10 10 10 1. Appendix D) clinical centrifuge and 4. in a single cell suspension of mouse splenocytes. we will use anti-CD4 or anti-CD8 antibodies and C'-dependent cytotoxicity to deplete all of the CD4+ or CD8+ cells.
1 100 100 100 100 100 100 100 TIB211 IgM (45% AS) 100 100 100 2.e. for FACS analysis the next day..0 0. Add 100 µl of the splenocyte suspension (i. 5. Wash the cells once more with an excess of DMEM-10% FCS and resuspend to 100 µl with DMEM-10% FCS.42 TIB211 supn't TIB211 IgM (30% AS) 100 100 100 100 10 10 1.5. Resuspend the cells to 100 µl in DMEM-10% FCS.1 10 1. 3. 4. then wash the cells one time with PBS and resuspend to 100 µl with PBS. 106 cells) and 100 µl of appropriately diluted antibody (10. and add 3 µl of FITC-labelled anti-CD4 and 3 µl of PE-labelled anti-CD8 IgG to each tube and incubate the cells on ice for 30 min. or TIB211 preparations) to each tube.0 0. Add 100 µl of 1% paraformaldehyde and fix the cells on ice for 30 min. and incubate the tubes at 37˙C for 30 min. Incubate the cells with the antibody for 30 min at room temperature. Wash the cells two times with DMEM-10% FCS (8 min at 1500 rpm in the clinical centrifuge). . Add 100 µl of the guinea pig complement to each tube. GK1.0 or 0. 6. Store the cells in the refrigerator overnight.1 µg protein from the HB121. 1.
3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells In this complementary exercise, we will learn how to use antibody-coated
paramagnetic beads that are specific for the isotypes of the anti-CD4 and anti-CD8 antibodies that you generated in §2.1 to purify all of the CD4+ or CD8+ cells, respectively, from a single cell suspension of mouse splenocytes. The real advantage of this system is that the selected populations are not killed or otherwise damaged by the isolation procedure, so that it may be possible to use them for functional studies after the purification procedure -- this is what we do in our lab to purify mast cells from tissues. The potential disadvantage is that in some cases the cell surface marker employed may well, by itself, activate or otherwise alter the physiology of the cells (e.g., anti-CD3 antibodies may activate T cells). We will confirm our depletion by using commercially available fluorescein-labelled anti-CD4 and phycoerythrin-labelled antiCD8 antibodies to stain the residual cell populations and then we will analyse the composite phenotype of these cells using two-colour FACS (fluorescence-activated cell sorter) techniques.
Materials single cell suspension of mouse splenocytes (at 1x107 cells/ml; Appendix D)
clinical centrifuge and 4, 15 and 50 ml polypropylene tubes Mini-MACS separation column (type MS; capacity 107 positive cells/column) Mini-MACS column magnet (Miltenyi Biotec Gmbh)
Reagents GK1.5 (anti-CD4 hybridoma) cell supernatants, purified IgG (§2.1)
TIB211 (anti-CD8 hybridoma) cell supernatants, ammonium sulfate ppt. IgM (§2.1) PBS (pH 7.2), containing 5% FCS & 2 mM EDTA (PBS/EDTA) mouse anti-rat IgG-conjugated paramagnetic beads (Miltenyi Biotec) mouse anti-rat IgM-conjugated paramagnetic beads (Miltenyi Biotec) fluorescein-labelled anti-mouse CD4 IgG antibodies phycoerythrin-labelled anti-mouse CD8 IgG antibodies
METHOD 1. Label and set up a series of tubes to receive reagents as follows: 2. Add 1 ml of the splenocyte suspension (i.e., 107 cells) and 250 µl of appropriately
diluted antibody (HB121, GK1.5, or TIB211; i.e., 25 µg) to each tube. Incubate the cells with the antibody for 30 min at room temperature.
Wash the cells one time with DMEM-10% FCS (8 min at 1500 rpm in the clinical centrifuge) and resuspend to 300 µl of PBS/EDTA. While the cells are washing, also wash the Mini-MACS column, flushing it through with the PBS/EDTA13. If air bubbles are present in the column, it may be necessary to back-flush the columns to get rid of the air bubbles. Mount the column in the magnetic holder in preparation for the separation in step 7 below. Add 25 µl of the anti-IgG paramagnetic beads to the GK1.5 tube from step 2 (i.e., 1:40 beads:cells), 25 µl of the anti-IgM beads to the TIB211 tube, and 25 µl of each to the no antibody and HB121 tubes, and incubate for 30 min at 6-12˚C (icewater bath). Wash the cells in PBS/EDTA, resuspending them to 300 µl PBS/EDTA. Apply the washed cells to the column (in the magnetic holder), and collect the flow through, then wash the column two times with an additional 1 ml (each time) of PBS/EDTA, collecting the flow-through each time. Pool the flow through cells, which comprise the marker depleted populations, and wash and count them. Remove the column from the magnet, clamping it to a ring stand, and flush the bound cells out of the column with 2 ml of PBS/EDTA, using the column plunger to assist in this operation, and collecting the eluted cells in a sterile tube.. Wash and count under the hemocytometer the retained cell populations. You will use these counts to compare with those obtained by FACS analysis of the total spleen populations (no Ab treatments). Stain the unselected populations from step 6 with the FITC-anti-CD4 and PE-antiCD8 antibodies at in §3.1, and fix with paraformaldehyde for FACS analysis the next day.
The first 10-20 drop to elute from this wash step will appear quite cloudy (due to elution of matrix residue). You should continue washing at least until the cloudiness of the eluting buffer diminishes to background.
3.3 Assessment of T cell proliferation (Blast assay) One of the basic tests in immunology is that of confirming that T cells are
responding to antigens as they should, or determining the precise levels at which these cells respond to antigens (specific immunoreactivity of the cells) or mitogens (overall responsiveness of the T cells). Traditionally, immunologists set up the target T cell population (e.g., PBL mononuclear cells, lymph node or spleen cells) in tissue culture, challenged the cells with the antigen of interest and then measured the proliferation of the T cells three days later by ascertaining their uptake of a radiolabelled DNA precursor (e.g., the nucleotide 3H-thymidine). This is a very sensitive assay, but it calls for the use of equipment and facilities specialized for use in handling radioisotopes. More recently, many labs have been using the MTT assay, performed exactly as outlined above for the LM-1 (§IL-1 ASSAY) and 7TD1 (§IL-6 ASSAY) cell proliferation assays, to accomplish the same goal. In this assay, we will examine the overall responsiveness of splenic T cells from BALB/c mice, by challenging the splenocytes with the T cell mitogen concanavalin A (Con A). In order to optimize the system however, we will need to determine the optimal concentrations of spleen cells needed for the assay, as well as the optimal doses of Con A required to induce proliferation. As with most immune responses, too much or too little of either can be inhibitory to the responses we wish to examine.
Materials humidified CO2 incubator
96-well tissue culture plates micropipetters, tips multi-channel pipetter clinical centrifuge, tubes 15 ml 15 ml centrifuge tubes hemocytometer ELISA plate reader (with a 595 nm wavelength filter)
Reagents BALB/c mouse splenocytes (5x107 nucleated cells/ml) in DMEM-10% FCS
DMEM-10% FCS media Concanavalin A (our stock is a 4 mg/ml solution in DMEM)
In one 96-well plate. and then add 20 µl of appropriately diluted ConA to each well (i.5 178 175 170 160 130 80 30 2 5 10 20 50 100 150 20 20 20 20 20 20 20 0.25 0.5 µg/ml. For the cell numbers titration portion of the experiment..5 1.5x106 cells/well.46 MTT 5 mg/ml in PBS) acidified isopropanol METHOD 1.0 2.0 7. total well volume of 200 µl). 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 cell number-response (cells x106 well) cell numbers titration ConA dose-response (µg ConA/ml) medium control CELL NUMBERS TITRATION [CELL] (x106/well) medium (µl) cells (µl) ConA (µl) ConA DOSE-RESPONSE CURVE [ConA] (µg/well) medium (µl) cells (µl) ConA (µl) 0.0 2.1 0.1 0.5 5 10 130 130 130 130 130 130 130 50 50 50 50 50 50 50 20 20 20 20 20 20 20 2. .e. Add sufficient DMEM-10% FCS to each well to bring the well volumes to 180 µl.5 1. Place the plates in the 37˙C CO2 incubator for three days. For the ConA dose-response curve. set up both cell number-response and Con A doseresponse curves using the plate format indicated below.25 0.5 5. and the volumes indicated in the table. use a splenocyte concentration of 2. use a ConA concentration of 2.
Remove 150 µl of medium from each well. At the end of the three days. examine the cells in each well to get a feeling for how the cells have responded to the ConA (strong ConA mitogenic responses will have induced cell clumping).90 min.4 min.SEM) for each treatment group. add 20 µl of MTT solution (5 mg/ml) to each well and return the plates to the incubator for 45 . 5. 4. .47 3. Calculate the mean OD595 (+/. and then read the plate at 595 nm wavelength on the ELISA plate reader. taking care not to also aspirate cells from the bottom of the wells. Next. Add 100 µl of acidified isopropanol to each well and place on the ELISA plate shaker for 3 . perform the statistical analyses and plot your data using bar graphs.
. Lyse the residual splenic red blood cells by hypotonic or ammonium chloride lysis (§5. or 120 µl of the splenocyte suspension. 30 µl of the 20% SRBC suspension (i. 2. 4x107 cells) either intravenously or intraperitoneally into 8-10 week old mice. as appropriate to bring the final volume of each tube to 300 µl. washed in PBS14 20% (v/v) SRBC in PBS/10%FCS syringes & 27 or 30 ga needles mice PFC assay chambers methoxyfluorane (anaesthetic) Reagents PBS H20 & 10x HBSS for hypotonic lysis of spleen RBC guinea pig serum diluted 1:2 in PBS/10% FCS Method 1. or 120 µl of RPMI-10%.4 Plaque Forming Cell (PFC) assay for IgM-producing cells Materials sheep red blood cells (SRBC. 30 µl of diluted guinea pig serum and 240.4. 60. 210. Inject 0. and generate single cell suspensions from their spleens. in PBS/10% FCS). 14 As an estimate of the numbers of SRBC available from the peripheral blood of a sheep. 1 ml of heparin-anticoagulated peripheral blood can yield ≈1x1010 red blood cells after 5-6 washes with PBS . Euthanize the mice after 4 days if they were immunized intravenously (or after 5 days if they were vaccinated intraperitoneally). and thoroughly mix the contents of each tube. 180. 2x108/ml).e.e.48 3. To a series of labelled eppendorf tubes.2 ml of the SRBC suspension (i.8) and bring the nucleated cells to a final concentration of 5x106 cells/ml 3. 30.. 4. Load 80 µl of the mixture into each assay slide chamber (see diagram below) and seal each with wax. add either 0.
6.5 h at 37˚C. Incubate the chambers for 1 .49 lower to appose against bottom slide microscope slide (2) double-sided tape gaskets (3) assembled double-chamber apparatus splenocyte-SRBC mixture 5. .1. Remove the chambers from the incubator and count the numbers of plaques of red blood cell lysis under the microscope.
5 ELISPOT assays for single cytokine.or Ab-producing cells A very powerful method to assess the abilities of animals to produce antibodies (Ab) or cytokines in response to antigenic stimulation is the ELISPOT assay. Th1 or Th2) of the mice to these immunogens. harvest cells on dy 21) . . 200 µl of 1% SRBC) pipettes/pipettors clinical centrifuge.05% Tween 20) streptavidin-alkaline phosphatase conjugate (strep-AP. to each well of the plate.: dy 1. Precoat each of the ELISPOT plates with capture antibodies or antigen (this can be done several days in advance).SRBC (i..v. Thus.: on dy 1 and dy 14. add the purified anti-IL-4 or anti-IFNγ antibodies in ELISPOT coating buffer at a concentration of 1 µg/ml or the ovalbumin at a concentration of 5 µg/ml. dy 14.1% SRBC. diluted 1:5000 in PBST) METHOD 1. Cover and seal the plates with parafilm and incubate overnight at 4°C.4) Lymphocyte separation medium ovalbumin (5 µg/ml coating buffer stock solution) PBST (PBS with 0. 15 ml tubes 96-well ELISPOT plate Reagents anti-mouse IL-4 and anti-mouse IFNγ capture antibodies (1 µg/ml coating buffer stock) bromo-chloryl-indoyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate biotinylated anti-mouse IL-4 and anti-mouse IFNγ antibodies (detection antibodies) DMEM-10% FCS ELISPOT blocking solution (DMEM-10% FCS) ELISPOT coating buffer (carbonate/bicarbonate. To do this. This procedure is used to detect the abilities of individual B or T (or other) cells to secrete their products. Materials splenocyte suspensions from mice vaccinated with: . pH 9.50 3.ovalbumin/alum (i. 200 µl of 0.p. We will assess the abilities of splenocytes from BALB/c mice that have been immunized with ovalbumin-alum to produce ovalbumin-specific antibodies of varying isotypes or those of these mice or SRBC-vaccinated mice to produce IL-4 or IFN-γ in response to antigenic challenge in vitro.e. we will in effect by phenotyping the CD4+ T cell responses (i.
G2a. G1. IgE antibody ELISPOT 1 A B C D E F G H 2 3 4 5 con't 6 7 8 9 10 11 12 con't no IFNg no cells no secondary 1x10e6. F no SRBC anti-IFNg.M.G2a. 1.G2a.10x10e51. with Ag.5. no Ag.10x10e5. G1. no secondary C D 1. IgM. +SRBC 1. no SRBC anti-IFNg. anti-IFNg + ovalbumin E anti-IL-4.G2a.5. anti-IFNg.5. no biotin Ab 5x10e5.10x10e5.M 1x10e6 cells with Ag.10x10e5 (OVA) anti-IL-4.M no a-IL-4 no cells no cells.10x10e5 1. no Ag. no secondary no Ag.anti-IL-4. G1.10x10e5 anti-IL-4.5. G1. 1. with Ag. 1.10x10e5. -IFNg ELISPOT B OVA mice anti-SRBC mice A anti-IL-4.10x10e5. G no anti-IL-4 +SRBC no blocking no anti-IFNg H no cells anti-SRBC mice 1 2 12 3 4 5 6 7 8 9 10 11 .G2a.5.5.M anti-IFNg. G1. with Ag.M 1x10e6.51 A anti-OVA IgG1.5.E 1x10e5. with Ag.5. no secondary no cells anti-IL-4 B + ovalbumin. no blocking 1. IgG2a.
for it will reduce the assay background substantially. The next morning. Add 100 µl of biotinylated anti-cytokine or anti-isotype antibody (diluted to 1 μg/ml in PBST) to each well. add 100 μl of diluted strep-AP to each well. 0. Do not to disturb the plates while they are incubating. so that each cell secretes all of its cytokine/antibody in only one location.e. . but do not touch the bottom of the wells. Remove the ELISPOT plate from the incubator and dump out the blocking solution. use 3.and SRBCsensitized mice. add 22 μl BCIP. Add 100 μl of freshly prepared BCIP/NBT substrate to each well. 6. Remove the blocking solution. 10. 3. remove the cells from the plate by first agitating the plates on the ELISA plate shaker for ≈3 min.5).0% suspension) to each well. Repeat this wash procedure 6 times. remove the capture antibody from the wells by inverting the plate and sharply flicking it. Incubate the plates at room temperature in the dark until spots develop or the plate background begins to increase (approximately 30-45 minutes). mix by inverting and then add 16. Wash the plates 5 times with PBST as in step 6.05 M MgCl2. Generate a single cell suspension from the spleens of an OVA. Add 100 μl of spleen cells and 100 µl of antigen (ovalbumin @ 2. Incubate the plates at 37°C for a minimum of 1 hour (or until the cells from the next step are ready). 4.5 µl BCIP & 12. and incubate the plates at 37 °C for 8 hours. for smaller volumes..75 ml buffer. and incubate the plates overnight at 4°C. and flick the PBST out as above. 7.5 h at room temperature. flicking and banging on paper towels). Continue washing the wells with 200 μl of PBST -vigorously pipette the contents up and down. 8. and incubate the plates for 1.5 μg/ml or SRBC @ 1. 9. and then inverting them and vigorously flicking the contents from the wells. After 8 h. 16. Remove the excess PBST by banging the plate upside down on paper towels. 0. 5. rinse as above and add 100 μl of fresh blocking solution to each well. each time removing all of the H2O from each well as above (i.5 μl NBT and again mix. Wash the plates 10 times by repeatedly dunking the plate in a large beaker of distilled-deionized H2O. Rinse out each well with 200 µl of blocking solution by pipetting the solution up and down several times with a multichannel pipetter (do not touch the bottom of the well with the pipette tips!). (To 5 ml of 0.52 2. Careful washing is important. and resuspend the cells to 1x107 cells/ml DMEM-10% FCS.37 µl NBT).1 M NaCl.1 M Tris (pH 9.
Count the spots under low magnification under the dissecting scope. Allow them to dry overnight (with the lids off). 12. as the background fades dramatically when the plates dry. or using an image analyser .53 11. flick out the substrate and wash the plates 3 times in H2O by the dunking method. To stop the reaction.
There is very little difference between the ELISPOT and ELISA assays .000. ATCC MAb # HB121 (mouse IgG2a anti-human IgE) and ATCC MAb (mouse IgE anti-DNP) ascites fluids work well for the antibody ELISA standards.g. the wells are coated with the non-antigenspecific immunoglobulin standards or the antigen in question. and IgE standards (commercial or appropriate hybridoma supn't15) 15 ATCC MAb # (mouse IgG1 anti-human IL-8). one can use high antibody value reference sera generated by vaccinated of mice with the antigen of interest. It can detect many antigens with a sensitivity of ≥1 pg/ml. While the ELISPOT tells you how many cells are secreting the antigen being detected.. using ascites fluid dilutions ranging from 1:50 > 1:1. Alternately. the latter should capture the antigen-specific antibodies from the samples. the wells are blocked with a protein solution that is not recognized by the capture antigen or standards (e. from ova-vaccinated mice) ovalbumin for capture of antigen-specific antibodies IgG1. and then the biological samples are applied.g. Subsequently. IgG2a. For example..54 3.the ELISPOT detects cytokine or antibody production in situ by viable cells while the ELISA detects proteins which are present in a soluble form in biological fluids. DMEM-10% FCS or 1% bovine serum albumin). The captured antigen-specific antibodies are then detected precisely as are the cytokines/antibodies in the ELISPOT assay (§3. the ELISA will quantify the product precisely. The former assay is performed in nitrocellulose paper-lined 96-well plates and utilizes a precipitating indicator dye (so that individual cell traces are detected). and then blocked. although for many others sensitivities of 100-200 pg/ml are difficult to achieve. while the latter is performed in high protein-binding plastic 96-well plates and employs soluble indicator dyes. 3.6 ELISA ASSAYS (Enzyme-linked Immunosorbent Assay) The ELISA assay is a powerful method for the detection of specific antigens (be they from pathogens or those specific for antibodies or cytokines).000.1 ELISA assay for detection of antigen-specific antibodies In the antigen-specific antibody ELISA.6.5) Materials Immulon-4 ELISA plates pipettes/pipettors Reagents experimental sera or other putative antibody source (e. BALB/c mice produce a very strong IgE and IgG1 response following intraperitoneal vaccination (dy 0) and boosting .
(dy 14) with 5 µg of ovalbumin conjugated to 1 mg of alum. we have not "played" with the latter system sufficiently to suggest that it could not yield equivalent results under the correct circumstances. then cover the plate(s) and incubate overnight at 4°C. 4. 2. Add 200 µl of PBST to the antibody standard wells. IgG2a. . Dilute the biotinylated anti-Ig isotype antibodies to 0. Cover the plate and incubate overnight at 4 °C. 1 component substrate for SA-HRP) 1% sodium dodecyl sulfate (SDS. Coat the wells of the Immunolon-4 plates with the antigen to be used for antibody capture (ovalbumin @ 5 µg/ml in carbonate coating buffer) or with the diluted antibody standards. Wash the wells 4 times with PBST. 16 In our hands.5 µg/ml in PBST (the optimal concentration will need to be determined empirically). To do this. at room temperature for 2 hours. optional stop solution for reactions) METHOD 1. Cover and incubate at room temperature for 90 min. substrate for SA-AP) 2-2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 (ABTS. then fairly forcefully hit the plate upside down on a stack of paper towels to remove the excess fluid from each well. as appropriate. Add 100 µl per well. Sera or plasma from these mice can be used as reference standards.05% Tween 20) DMEM-10% FCS streptavidin-allkaline phosphatase (SA-AP) or SA-horse radish peroxidase (SA-HRP)16 3 mM p-nitrophenyl phosphate (in 0.05 M Na2CO3/0. Wash the wells 2 times with PBST. and to the ovalbumin-coated sample wells add 100 µl of the samples (diluted ≥1:50 in DMEM-10% FCS) to each well. then turn the plate upside down and flick the wash fluid into a sink. 3.05 mM MgCl2. as appropriate for each sample or standard. one can either use a squirt bottle of PBST or a multichannel pipettor to fill each of the wells and allow to stand for 30 60 seconds. the streptavidin-horse radish peroxidase/ ABTS enzyme/substrate combination gives vastly superior results to those obtained with the SA-AP/p-nitrophenyl phosphate enzyme/substrate system although. Block the plates by adding 200 µl of DMEM-10% FCS to each well and incubate. in all honesty. covered.55 biotinylated IgG1.5 .2. and add 100 µl of each per well. and IgE detection antibodies ELISA carbonate coating buffer PBST (PBS with 0. Wash the wells 2 times with PBST as in step 2.
making calibration of the results difficult or impossible. several hours or even overnight with particularly weak samples).45 min17 at room temperature. Wash the wells 8 . For the ABTS substrate. Wash the plates 8 .10 times with PBST. the plates can be left longer (e. However. . and add 100 µl of either SA-HRP (1:5000 in PBST) or SA-AP (1:5000 in PBST) to each well. 17 If the optical densities of the standards or samples does not come up to a useable level within this 45 min time frame. Add 100 µl of the ABTS substrate (SAHRP avidin-enzyme conjugate only) or freshly prepared 3 mMp-nitrophenyl phosphate substrate (SA-AP avidin-enzyme conjugate only) to each well. Read the plates using the ELISA plate reader. set at a reading wavelength of 405 nm.10 times with distilled H2O by the dunking method.. or stop solution may be added to reduce plate to plate variability when reading multiple plates. 6. Incubate at room temperature for 90 min.g. during this time.56 5. it is possible that the standards could become overdeveloped. the stop the reactions by adding 100 µl of 1% sodium dodecyl sulfate (SDS) to each well.. The plates can be read directly at this point. then place the plates in a dark location (e.g. a drawer works well) and allow the reactions to develop for 20 . 7. making sure to remove the excess fluid as above.
you can use a monoclonal in conjunction with a polyclonal anti-cytokine antisera. Thus.57 3.1. step 2).5 μg/ml in coating buffer. 2. covered. 1 component substrate for SA-HRP) 1% sodium dodecyl sulfate (SDS. the cytokine ELISA depends on using a cytokine-specific antibody which does not recognize the same cytokine epitope as the biotinylated detection antibody. and block the plate by adding 200 µl of DMEM-10% FCS to each well and incubate. Dilute the capture antibody of each pair to 0. Materials Immulon-4 ELISA plates pipettes/pipettors experimental samples for cytokine assay Reagents cytokine capture and biotinylated detection antibody pairs ELISA carbonate coating buffer PBST (PBS with 0.6. care must be taken to ensure than the antibodies/antisera employed will only recognize the cytokine of interest and that they will not inappropriately bind to immunoglobulin or other assay reagents and thereby give "false-positive" results. 3. as empirically determined. Wash the wells 2 times with PBST.6. Cover the plate and incubate overnight at 4°C. and add 100 µl of the standards or samples (diluted in DMEM-10% FCS) to each well. and add 100 µl to the appropriate wells of the ELISA plate. Wash the wells 2 times with PBST. as above (§3. Alternately. The precise levels of standards to use will depend on the detection limits of the antibody pairs employed. However.2 ELISA assay for detection of cytokines Rather than using antigen as the capturing agent. optional stop solution for reactions) METHOD 1. monoclonal antibody (MAb) pairs are required and are available routinely from many commercial sources. but will often .2. at room temperature for 2 hours.05% Tween 20) DMEM-10% FCS SA-horse radish peroxidase (SA-HRP) 2-2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 (ABTS.5 .
. The reactions can be stopped by adding 100 µl of 1% SDS to each well. set at a reading wavelength of 405 nm.5 . Add 100 µl of ABTS substrate to each well and allow the reactions to develop for 20 . 5. as appropriate. Wash the plates 8 . Wash the wells 4 times with PBST. making sure to remove excess fluid.5 µg/ml) in PBST and add 100 µl of each per well.10 times with PBST.58 cover the range of from 1 pg/ml to 1500 pg/ml. Wash the wells 8 . Cover the plate and incubate overnight at 4 °C. Cover and incubate at room temperature for ≈90 min. 6. Read the plates using the ELISA plate reader. Dilute the biotinylated anti-Ig isotype antibodies to an empirically-determined optimal concentration (usually 0. 7.45 min at room temperature.2. 4. and add 100 µl of the diluted SA-HRP (1:5000) to each well. Incubate at room temperature for ≈90 min.10 times with distilled H2O.
Set up the antigens for the intradermal skin tests in the 1 ml tuberculin syringes equipped with 30 ga needles. Lightly anaesthetize each mouse it turn (i.e. 5.7 In vivo assessment of T cell responses: Th1 versus Th2 responses Materials Ovalbumin. Trim away and discard the tissue from the outside of the ear. making sure that you keep it warm (use a heat lamp if necessary) and safe (from any overly dominant littermates) during recovery. 2. Replace the ISH fixative with ice-cold 70% ethanol and store the tissue in this solution at -20˙C until you are ready for tissue processing. euthanize one mouse from each group (i. two ovalbumin-sensitized mice and two SRBC-sensitized mice).and one ovalbumin-sensitized mouse) and take biopsies of the reaction sites. To do this. also set up a 15 ml tube (with cotton batting and methoxyfluorane) that you can use during the procedure.and SRBC-immune mice Tissue processor (for processing biopsies to paraffin blocks) 1 ml syringes and 30 ga needles scalpel blades Reagents Ovalbumin in Ca++/Mg++-free HBSS (40 µg/ml) SRBC in Ca++/Mg++-free HBSS (5% suspension) In situ hybridization fixative 70% ethanol METHOD 1. If this will take some time. one SRBC.. 3. use a fresh #12 scalpel blade to remove the ear and cut it in half longitudinally. Transfer each half of the ear into ice-cold ISH fixative and fix the tissue for 3 h on ice. so that it can be injected intradermally in the ear. away from the injection sites. The bevel side of the needle and the calibrated side of the syringe should be aligned if you are going to inject defined volumes based on the calibration of the syringe. .e.. 4. Return the mouse to its cage. Inject 25 µl of the 5% SRBC suspension intradermally into the right ear of the mouse and inject 25 µl of the 40 µg/ml ovalbumin solution intradermally into the left ear.59 3. At four hours post-injection and then again at 24 h. 6. 7. in order to keep the mouse anaesthetized on the bench. through the middle of the reaction (injection) site.
9. The processor will automatically process the tissue through to molten paraffin.60 8. Cut 6 µm paraffin sections of the tissues and dry them onto slides overnight at 42˙C.4. 10. so that upon sectioning you will obtain cross-sections of the tissue.1. Place the tissues into labelled tissue-tek cassettes and transfer into the tissue processor at the 70% ethanol step.2). 9. mount with permount and examine the tissues under the microscope. . 11. Embed the tissues in paraffin such that the ear tissues are standing straight up in the molds. Stain the sections with Giemsa stain using the stipulated protocol (§6.
.. then equilibrate to PBST buffer for 2-3'.5) BCIP/NBT (see §3.. 2. Run paraffin sections through two xylene baths (10' & 5') to remove the paraffin.8 Immunohistochemical detection of cytokines in tissues Immunohistochemistry is a powerful tool for the detection of single cells which are positive for any marker of interest for which suitable antibodies exist (e.5) METHOD 1.PBS containing 0. etc).5-positive CD8 cells. rabbit anti-TNF) biotinylated secondary antibody (e. The tissues or cells are perhaps best prepared in fixatives such as Bouins or Carnoys. then rehydrate the tissues by passing the slides through the graded ethanol baths (2x100%.g. GK1. § 3. It is thus similar to FACS analysis.5) commercial streptavidin-alkaline phosphatase (SA-AP. 90%. each < 1 min). we often use our in situ hybridization fixative (§5. In our laboratory.g. 100%. Incubate the rehydrated tissue sections in 10% normal goat serum for 2 h @ RT in order to block the non-specific binding capacity of the tissue immunoglobulin receptors (FcR) for the antibodies to be used subsequently. 90%. 50%. 50%) for hydrating tissue sections PBST .e. rendering them much less detectable using standard protocols...61 3. cytokine-secreting cells. biotinylated goat anti-rabbit IgG antibody. F4/80 antibody-positive MØ. Materials micropipetters ISH-fixed cell suspensions or paraffin-embedded 5-7 µM tissue sections Reagents xylene graded alcohol baths (i.g.3) for experiments calling for either in situ hybridization (ISH) or immunohistochemistry (IHC). allowing one to assess the prevalence of marker expression in situ. §3. 3. but has perhaps traditionally been used most often with fixed tissues or cells. 70%. Circle the tissue sections with wax pencil to reduce the amount of reagents required to saturate the tissue sections in each of the following steps.05% Tween 20 normal goat serum primary anti-cytokine antibody (suitable for immunohistochemistry. within the context of ongoing physiological or pathological processes. 70%. e. since formaldehyde fixation (especially prolonged fixation) can destroy the "antigenicity" of many epitopes.
Overlay the tissue sections with ≈75 µl of the biotinylated secondary antibody. Wash the tissue sections three times for 5' each in PBST. this time with SA-AP diluted to 1:5000 in PBST. 8. 7. generally for 2h @ RT.62 4. 9. Overlay the tissue sections again. Overlay the tissue sections with ≈75 µl of the primary antibody. using empiricallydetermined optimal concentrations of the antibodies (culture supernatants are often used @ 1:5. and monoclonal antibody ascites fluids @ 1:250-1:10. . again using empirically-determined optimal concentrations of the antibodies. 10. Wash the tissue sections three times for 5' each in PBST. and incubate for 90 ' @ RT to label the secondary antibodies in the tissue sections. Wash the tissue sections three times for 5' each in PBST. Overlay the tissue sections with the commercial solution of BCIP/NBT for 20 .000) ON @ 4C.40' @ RT (continue staining until the antigen of interest becomes apparent or until a non-specific general tissue background staining begins to appear). Transfer slides to H2O and counter-stain with a water-based counter-stain such as Gill's haemotoxylin (§ ) and cover-slip with aqueous mounting medium. 11. commercial preparations of purified antibodies @ 1:501:250.1:100. 5. 6.
tips. for mRNA species that are only weakly expressed. for electrophoresing and blotting it.63 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION : 4. test tubes micropipetters single cell suspensions of activated and control Cl. I use the former method for purifying large amounts of RNA and the latter for minuscule amounts of RNA -. Materials samples for RNA extraction (tissues or cultured cells) RNAse-free pipettes. but many people instead use an acidic phenol-chloroform extraction protocol. you will be examining the expression of the mRNA for the protein by electrophoretically separating them according to their size and confirming their identities with specific cDNA probes.there are an increasing number of very good mRNA purification kits available on the market as well. you will need to purify the mRNA from the total cellular RNA pool.1 Purification of cellular RNA While the protocol outlined herein is for the purification of RNA from cells. except that with tissues one uses a tissue disrupter or homogenizer. while we will not be isolating mRNA from the total cellular RNA (because our target mRNA species should be highly expressed).MC/C57. 4. 3 & 6 h) Reagents DEPC-H20 20% lauryl sarcosine 5. Finally. and for probing the blots.7 M CsCl/sodium acetate .1.1 cells (time course of 0. except that instead of examining the expression of proteins by electrophoretically separating them according to their size and confirming their identities with specific antibodies. it is essentially the same as that for tissues. We will be using CsCl gradients to purify the RNA. We will examine the methods for the purification of the mRNA.5 M GSCN lysis solution 5.1 Northern blotting Northern blotting is much like Western blotting.
and finally. using a 3 ml syringe. then add the sarcosine after the tissue homogenization. and taking care to avoid frothing the samples excessively. Fit the buckets onto the SW55Ti rotor. #1 & 4.1 min. The next day. For samples homogenized by vortexing only. 18 When extracting RNA from tissues. Wait for the rotor to come to speed to confirm that everything is operating smoothly. to within 1/100th of a gram. grind the tissues using a polytron-type homogenizer in GSCN without sarcosine (to avoid foaming). In place of a polytron to grind up the tissues. one could also use a mortor and pestle with liguid nitrogen to accomplish the same thing. needle. Load the tubes into the SW55Ti rotor buckets and balance the loaded buckets & caps in pairs (i. 8. and vortex vigorously for ≈30 sec . shear the DNA by forcefully aspirating and ejecting the lysate through an 18 ga. (When the DNA is shorn sufficiently. and the buckets from the rotor. and check the "O" rings and lubricate using vacuum grease. if necessary. pellet the appropriate tubes of C57 cells by centrifugation.64 METHOD 1. Precentrifuge the homogenized tissues for 30 min to 2 hours in the ultracentrifuge to get rid of any insoluble tissue remnants. maximum temperature to ≈30˙C. brake off. and fill the buckets only to ≈2 mm from the top (you will need to leave room for the addition of additional GSCN as required for bucket balancing (step 4). very carefully place the rotor on the centrifuge spindle. 6. Add 3 ml of GSCN lysis solution to each tube. timed operation. Pipette 1.e. The last few milliliters are best gotten by turning the tube upside 7. the sample will drip discretely from the tip of the needle rather than coming out as a viscous linear strand. run time to ≈20 h. Switch on or set the following centrifuge control options: vacuum on.. switch off the vacuum. #2 & 5. Take the tubes from the buckets. then generate a paste on the tube walls by vigorously flicking the tubes. on. and then gently overlay the CsCl with the cell lysates. and gently close the weighted door. run temperature to ≈22˙C. At each time in the activation time course. Do not allow the CsCl and samples to mix.0 ml of CsCl density gradient medium into each of 6 polyallomer ultracentrifuge tubes. wash them out with hot tap water & dry. Tighten the lids down .000 rpm. Completely aspirate the liquid contents of the tube using a Pasteur pipette attached to a vacuum apparatus.18 2. one drop at a time. being very careful not to damage the overspeed disc on the bottom of the spindle!. open the door and remove the rotor from the spindle. #3 & 6). 4. after the rotor has stopped. .) 3. rotor speed to 42.finger-tight only! 5. Use GSCN lysis solution to balance the buckets by adding it to each tube with a syringe fitted with an 18 ga needle. slow acceleration on.
transferring both washes into an RNAse-free 1.0 -. aspirate the supernatant again and leave the tubes open on the bench for ≈30 .the lower the ratio.5 are not uncommon and may still yield excellent results with Northern analyses). use quartz cuvettes) of each of the samples. . microfuge the RNA at 4˙C for 30 min. break up the RNA pellet with the tip of the eppendorf tip. take the calculated volume required for 20 µg and transfer it to a new RNAse-free eppendorf tube. Re-microfuge for ≈15 min at 4˙C. Remove 5 µl of the RNA from each tube and transfer to another tube containing 995 µl of H2O. then you will need to concentrate the samples.5 ml eppendorf tube. 11. Store at -80˙C. resuspend the pellet in the required volume of H2O or Northern blot RNA sample prep buffer and dissolve at 65˙C.8 mm across.1 volumes of 3M sodium acetate (pH 7. If your RNA solution is too dilute for subsequent Northern analysis (e. and dissolve the pellet in ≈50 . 10. Add 0. 20 µg represent >≈30 µl of RNA).5 volumes of 100% RNAse-free ethanol. The pellet will be anywhere from 1 . multiply the OD260 by 10 to get the concentration in µg/µl (i. an OD260 of 0. Calculate the OD260:OD280 ratio and the concentrations of the stock RNA solutions as follows: The 260:280 ratio should be between 1. carefully aspirate the supernatant (avoid the minuscule pellet!) and wash the pellet with ≈500 µl of ice-cold 70% ethanol. If necessary. Immediately transfer the stock tubes of RNA to dry ice and then to the -80˙C freezer.5 and 2. or on dry ice. the more protein contamination but.108 would mean that the stock RNA concentration was 1.08 µg/µl. ratios of 1. To calculate the concentration of the stock RNA.g. Close the tube and transfer it to a 65˙C H2O bath for ≈5 min to completely dissolve the RNA.. cut the tubes off about 1 cm above the bottom using a heated scalpel blade. The next day. practically speaking.. To do this.0 (pure nucleotide solutions have an OD260 of 2. down to allow the fluid to drain to the pipette. as above.400 µl of DEPC-H2O (depending on the size of the pellet) by repeated vigorous pipetting. One at a time.e. Stay away from the gelatin-like pellet of RNA at the bottom of the tube.08 x 300 = 324 µg of RNA). then briefly microfuge to sediment the contents. repeat the H2O step to make sure you get all of the RNA. until ready to use.0) and 2. Determine the optical density (260 nm and 280 nm wavelength. for a 300 µl solution.60 min to dry.65 9. vortex briefly to mix and put in the -80˙C freezer overnight. When the ethanol has dried. that would mean that you had purified 1.
2% agarose gel 10 mg/ml ethidium bromide 0. Materials RNAse-free horizontal gel electrophoresis gel apparatus & power-pack Zeta-bind transfer membrane (or other) RNAse-free glass or plastic pipettes micropipetters & tips filter paper (e. Incubate 2. pg 94) to a depth of about 8-10 mm (since the RNA samples will be negatively charged and will migrate towards the positive electrode.g.03% methylene blue in 0. Wash the Northern blotting apparatus with DEPC-H2O.3M ammonium acetate RNA sample prep solution (for denaturation of RNA prior to electrophoresis) RNA sample dye/loading solution (for visualization of progress during run) METHOD 1.66 4.2% agarose/formaldehyde/MOPS gel (appendix . add ≈15 µl of the RNA sample prep buffer and ≈2 µl of RNA load buffer/dye.1. a protocol that often depends heavily on the use of RNAses.. and then pour a 1. Whatman #1) & absorbent paper (e. remove the comb from the gel and fill up the chamber with 1x MOPS running buffer. . covering the gel to a depth of ≈2-3 mm with buffer. When the gel is fully solidified (this can be expedited by doing the last of the cooling step in a refrigerator). it should be reserved for RNA work and should not be used for preparation or analysis of plasmids. 10xSSC 1. paper towels) Reagents agarose DEPC-H20 ammonium acetate formaldehyde 5X MOPS.20 µl of H2O.2 Electrophoresis of RNA & transfer to membranes It is critical that the electrophoresis apparatus used is more-or-less free from RNAse contamination. make sure that you position the gel with the wells closest to the negative electrode)..g. Therefore. To eppendorf tubes containing 20 µg samples of RNA in a volume of 10 .
67 3.75% down the gel. and then apply several more pieces of filter paper. (The leading dye front will migrate just in front of the 18s rRNA. Run two sets of samples. 4. cut some large pieces of parafilm membrane and place them around the gel. for the portion to be stained with ethidium bromide. Assemble the northern blotting apparatus as follows: Cut a wick of 4 . 6. For the half of the gel to be transferred to the nylon membrane. cut to the size of the membrane. and cover this whole construct with ≈4-5 inches of paper towel.5 layers of Whatman #1 filter paper that can run the full length of the gel apparatus gel platform and extend down into the fluid reservoirs. being careful to not puncture the very fragile bottoms of the wells with the pipette tips. while the trailing dye front will migrate just behind the 28s rRNA in each sample. Load the RNA samples into the wells in the gel. follow alternate steps 5a . a 500 ml reagent bottle that is ≈ half full) and secure in place. and incubate the gel in the water bath for 20 min with gentle rocking. Equilibrate the half for transfer to 10xSSC. Apply a piece of the Zeta-bind membrane (cut the overlap the gel by a few millimeters) directly to the gel. When all the samples are loaded.7. so that the transfer buffer can only wick into the paper towel by going through the gel. . Finally. by incubating in 10x SSC for 20 min with gentle rocking.) Remove the gel from the gel apparatus and carefully cut it to separate the Northern blotting portion from that destined for ethidium bromide staining.g. Place the gel. which will absorb the buffer that wicks through the gel and Zetabind membrane. run the gel until the leading dye front is approximately 50 . The portion to be stained with ethidium bromide should be incubated for another 20 min in H2O..6a. then briefly pulse microfuge the tubes and hold on ice until ready to use. making sure that there are no air bubbles trapped underneath. one for Northern blotting. upside-down onto this wick. Next. directly on top of the transfer membrane. follow steps 5 . Transfer both 'halves' into DEPC-H2O. the samples in a 65˙C water bath for 10 min. and a separate set for staining with ethidium bromide (for confirmation that the RNA samples are intact). 5. place a weight on top of the paper towel (e. 5a.
03% methylene blue in 0.1 M ammonium acetate solution for another 20 min. then destaining the blots in DEPC-treated distilled water for ≈2 minutes. Assess the integrity of the RNA and the efficiency of transfer by staining the blots in 0. but if necessary the background can be decreased by further washing with water. To the portion of the membrane to be stained with ethidium bromide.68 paper towel filter paper nylon membrane gel for blotting filter paper wick 10x SSC gel/blotting apparatus 6a. the RNA is completely stable. (Stored in this manner. For the portion of the gel that was blotted. as well as a subtle smear of mRNA that runs from above the 28 s rRNA band to below the 18 s rRNA band.) 8. if required).1 M ammonium acetate containing 0.3M sodium acetate (pH 5. If the RNA is intact and transferred efficiently. you will readily see the 18s and 28s ribosomal RNA bands on the blots. Stain the gel for ≈45 min and then examine under UV light. then some RNA degradation has taken place – the extent of the degradation can be judged readily in this manner.2) for 45 seconds (or more. (If the rRNA bands are not crisp and sharp looking. photograph the gel as is. they can be successfully probed up to several years later). . Place the damp blot on damp filter paper into the irradiation apparatus and set it to automatically deliver the correct energy to the membrane (auto-crosslink).5 µg/ml ethidium bromide. such that the blots can be stored indefinitely at room temperature or in the freezer. transfer the gel into 0. and then into 0. disassemble the transfer apparatus the next morning and bind the RNA to the membrane by UV cross-linking using the Stratalinker. 7. After cross-linking. If the background is not too high.
69 4. Incubate the reaction mixture overnight at room temperature (or for >3 h @ 37˚C).1. 2. Follow the isotope using a Geiger counter. 3. NTPs/random hexamer soup). but not the elution tubing. Mix together 500-1000 ng of purified cDNA and H2O. Purify the 32P-labelled cDNA from the unbound probe by column chromatography (see accompanying diagram). Thus the Geiger counter will detect all of the elution of label from the column (both that incorporated into the cDNA and that still unincorporated). masking from the Geiger counter the column itself and the collection vessel. 4. . METHOD 1. using the random hexamer method. and 1 µl of Klenow fragment. then denature the cDNA by heating to 90˚C for 10-15 min.e. In this method a solution containing essentially all possible nucleotide hexamers is used to prime the synthesis of secondary stands of cDNA from the denatured primary strands in a reaction in which one of the component deoxynucleotides is 32P-dCTP. Run the 50 µl reaction mixture over an ≈8 ml Sepharose G-50 column. 5 µl of 32P-dCTP. carefully chasing the 50 µl reaction into the matrix with 2 sequential ≈1 ml aliquots of STE buffer. 3000-8000 Ci/mmol) Oligolabelling kit (Pharmacia) containing the random hexamer primers and the Klenow fragment of DNA polymerase. Materials cDNAs for each mRNA of interest Plexi-glass 32P-energy-blocking safety shield glass pipettes plastic wrap Geiger counter Reagents 32P-labelled dCTP (Mandel-New England Nuclear. to a final volume of 34 µl.. Transfer the cDNA to a 37˚C incubator for 5 min.3 32P-labelled cDNA probe synthesis The probes used to detect the mRNA species of interest are generated by in vitro labeling of individual cDNAs specific for each mRNA. and eluting the labelled cDNA with STE. The labelled cDNA is then purified from the unbound dCTP by chromatography through Sephadex G-50. Add 10µl of the oligolabelling reaction mixture (i.
etc. . so that the labelled probes cannot be stored for any more than 3 days to a week without decaying beyond usefulness. for 32P contamination. tube or simply do not elute it from the column. making sure to monitor all pipettes. then aliquot and freeze the labelled material until ready for use. discarding it instead with the column and matrix. 7. shields benches. 8.5x107 cpm of 32P-cDNA. is in the column elution tubing).e. Either collect any residual activity into a second. Wipe test all surfaces and equipment to confirm your Geiger counter survey and clean up any remaining contamination using a detergent such as Count-Off (New England Nuclear). Calculate the volume of column eluate required to yield 1. Enter your wipe test results in the lab log books.70 poly-prep column Seph G-50 unincorporated 32P beta energy shield (with window @ elution tubing) beta energy bound 32P elution tubing gieger counter collection tubes 5. has eluted.19 Clean up your work area. 19 32P is a highly unstable isotope. 6. Determine the radioactivity present in the labelled cDNA by adding a 5 or 10 µl aliquot of the cDNA to 4 ml of liquid scintillation cocktail and counting it in a β counter. continuing to do so until this first peak of labelled material. which comprises the 32P-cDNA in its entirety. discard. Once labelled material begins to read on the Geiger counter (i. begin to collect all of the eluate into one new tube..
& heat-sealing unit) glass pipettes plastic wrap Geiger counter Reagents 0.1x SSC/0.5x107 cpm of 32P-labelled cDNA probe (≈250 .71 4. 4. and washing (Zeta-Bind nylon membranes) Materials Plexi-glass 32P-energy-blocking safety shield U. TNFα & actin) METHOD 1.2x SSC/0.1% SDS pre-hyb/hybridization solution 32P-labelled cDNA probes (TGFβ.1. 5. hybridization. Block the non-specific 32P-cDNA-binding sites on the membranes by incubating the blot in the hybridization oven for 60 min at 65˙C in ≈15 ml of 0.5% SDS 2x SSC/0.. irradiation apparatus (e. Stratalinker) or vacuum oven rotary hybridization oven with hybridization tubes (or water bath. Next. The probe must be boiled before addition to the blot to denature the double-stranded DNA (and allow the anti-sense strand to hybridize to the mRNA on the blots).V. and should not be allowed to cool before addition to the blot. Remove the Northern blot blocking solution from the blot and replace it with ≈15 ml of hybridization solution. add 1. 2. and rinse the bottles twice for 5 min .1X SSC/0.1 % SDS 0. The next day. Pre-hybridize the blots in this solution for 3 h to overnight at 42˙C. cross-link the RNA onto the membranes either by U. remove the very radioactive hybridization solution from the hybridization bottles (discard in the 32P-liquid waste canister). Incubate the blots with the probe again overnight at 42˙C. After transfer of the RNA from the gel to the Zeta-bind membrane.4 Pre-hybridization. irradiation or by baking the blot in a vacuum oven (50˙C for 4 h). making sure that the oven rotator is operating.V. heat-sealable bags.1000 µl volume) to the 15 ml of hybridization solution containing the blot. 3.g.5% SDS.
.50 ml of 0. and discard the spent washings into the 32P-liquid discard canister. If the counts appear to be specifically bound. each with 2xSSC/0. of the radioactivity is associated with bands at the expected position (molecular mass) on the blot.1% SDS. 7. do not allow the blot to dry!).72 6. most of the time. discarding the spent washing into the 32P-liquid discard canister.1% SDS to each bottle and incubate for 30 min at 42˙C. increasing the stringency of the washes (i.e.1% SDS wash is adequate. Using the Geiger counter. If it is not.2xSSC/0..2xSSC/0. Remove the blot from the bottle and lay it flat out on a piece of plastic-wrap. Add 25 . as drying will permanently fix all of the (i. specifically and any residual non-specifically bound) 32P-labelled cDNA probe to the blot. 60 min at 42˙C in 0. decreasing the SSC concentration &/or increasing the wash temperature) but.. scan the blot to determine whether most. wrap the blot completely in plastic wrap and either store it in a freezer until ready to proceed or place in the autoradiography exposure cassette (again. then you need to continue washing the blot(s). Do not allow it to dry at this point. Repeat this 30 min/42˙C wash a second time. if not all.e.
and then adjust the time to get an optimal signal intensity. unless processing by hand METHOD 1. but if it is not easily detectable.3 weeks. 4.e.e. such that their shiny surfaces face one another. You may need to do a short exposure to get a feel for the intensity of the signal. place two 'enhancing screens' into the cassette on top of each other (as in a sandwich). perform a densitometric analysis of the band . then a 1 .5 Detection of mRNA bands by autoradiography Materials autoradiography exposure cassettes ('enhancing screens' highly desirable) Kodak X-OMAT AR x-ray film automated x-ray film processing unit (or hand processing equipment/solutions) Reagents none. remove the film from between the enhancing screens and feed it into the x-ray film processor. In the dark-room. If the signal is readily detectable with the Geiger counter. Place the probed. §NORTHERN BLOTTING: Pre-hybridization. Place the exposure cassette in a -80˙C freezer until you are ready to develop the film. under safelight illumination. and High-stringency Washing]). Hybridization. Under safelight illumination. and then close and lock the cassette.. remove the cassette from the -80˙C freezer and allow it to come to room temperature (this avoids condensation on the film later on). clamp sheets of plywood or plexiglass to the outside of the cassettes to hold all layers of the assembled exposure apparatus tightly together. then the exposure time may need to be extended to 1 . open the cassette. plasticwrapped blot in the cassette. Do not turn on the lights or exit the processing room until you are sure that the film has completely entered the processor (usually there is a signal from the machine). not in contact with the blot). 2. place a sheet of x-ray film inside the enhancing screen 'sandwich' (i. 3. For development of the film. 5. Provided the mRNA signals on the blot are not over-exposed (i. For the much less expensive 'leatherette' cassettes..3 day exposure is usually adequate. In the laboratory. have not photographically saturated the film). outside of the enhancing screen sandwich. The length of time will vary depending on the intensity of the specific mRNA signal (roughly determined with the Geiger counter [see step 7.73 4.1. and for autoradiography cassettes without built-in 'enhancing screens'.
g.74 intensities of each mRNA band. .g. TNFα). Determine the relative signal intensities of each experimental mRNA band by expressing it in terms of the relative amounts of actin mRNA in each lane (ideally.. actin) and the experimental mRNA (e. it is because you loaded different amounts of RNA in each lane). for both the housekeeping control mRNA (e..if they do. the actin signals will not vary between lanes -.
5 mCi/ml.2 In Situ hybridization (ISH) In the experimental procedure that we will follow.1 cells for the expression of TNFα and TGFβ. Thus we will be probing cytocentrifuge preps. briefly fixed again to neutralize any RNAse activity. Synthesize the cRNA probes by transcribing the cDNA in the linearized in vitro transcription vector (i. and finally... The tissues are probed and washed at a relatively high temperatures to prevent non-specific annealing of the cRNA probe to other tissue mRNA species. For each experiment you will need to generate both sense and anti-sense (reciprocal transcriptional orientation) . 4. The cells are fixed in ISH fixative for as short a period as is consistent with good cytoarchitecture.75 4. we will probe activated Cl.MC/C57. are hydrolysed in HCl and digested with protease to make the mRNA more available to the cRNA probes. but its sensitivity is perhaps one-tenth of that of Northern blotting. using 35S-labelled TNFα sense and anti-sense and TGFβ anti-sense cRNA probes. T3 or T7 RNA polymerase recognition sites upstream of the cDNA sequences of interest). the mRNA-bound 35S-cRNA probes are detected at the cellular level by dipping the slides in a liquid film emulsion and performing in situ autoradiography.e.e. and blocked with acetic anhydride to reduce non-specific sulfhydryl bond formation between the 35S-labelled probe and the tissues. ISH is extremely specific. a vector with SP6.2. thio-UTP) DEPC-H2O linearized template (i. in vitro transcription vector containing cDNA) METHOD 1. inasmuch as you can detect and identify individual cytokine mRNA-positive cells.1 Probe synthesis & purification Materials micropipetters & tips RNAse-free eppendorf tubes Reagents in vitro transcription kit or reagents RNAid RNA purification kit 35S-UTP (12. 1000 Ci/mmol) uridine 5'[α-thio] triphosphate (or.
7.5 µl total volume Briefly vortex the tube. spinning too long will pack the glass into too hard a pellet for subsequent dispersal) and remove and discard the highly radioactive supernatant using a 1 ml pipetman set at 500 µl.. 4. . which you then transfer into an empty scintillation vial (also labelled A. To do this add.e. for each probe to be prepared add the following ingredients (in order) to a 1. Purify the 35S-UTP-labelled cRNA from the reaction mixtures using the RNAid RNA purification kit. To accomplish this.0 µl template (linearized plasmid) 2.0 µl 1M DTT 0. 2 µl yeast tRNA 1 µl DNAse 1 µl RNAsin briefly vortex the tube and sediment the liquid by a ≈3" pulse-spin incubate the reaction tubes for an additional 15 min @ 37˚C.0 µl appropriate RNA polymerase 10. CTP.1 µl 5X transcription salts buffer 1. Pulse-spin the tubes for 4 counts (i.0 µl 35S-UTP 1. vortex. Remove 1 µl of each reaction mixture and dot it onto a filter paper disc (labelled A).5 µl RNAsin 0. Add 400 µl of RNAid kit wash solution to each tube and resuspend the pellet by repeatedly vigorously expelling and not too vigorously aspirating the 400 µl wash solution with a P200 micropipetter set at 150 µl (too vigorous an aspiration action 3. and place in a 37˙C H2O bath for 90'.4 µl nuclease-free H2O briefly mix the ingredients before adding the template (the spermidine in the 5x salts could precipitate the DNA in the template if not thoroughly dispersed) 2. and 250 µM UTP] 1. Remove the DNA template from the reaction mixture by digestion with RNase-free DNase I. 6. 5. pulse microfuge. and pulse-spin as above. Add 300 µl of RNA-binding salt to each tube and vortex. ≈4 sec. Incubate the tubes for 5 min @ room temperature to allow the RNA to bind to the scintered glass.5 µl NTP cocktail [10 mM @ ATP. 2. for ß-counting. Add 86 µl DEPC-H2O.76 probes.5 ml microcentrifuge tube on the benchtop 2. Add 2 µl RNAid-kit 'glass milk' and vortex each tube to disperse the 'glass milk' evenly. below). GTP.
11. and place the tubes in a 55˚C water bath for 3 min.e. and remove and discard the highly radioactive supernatant using a 1 ml pipetman set at 500 µl. 13. 12. ATP. cap them securely and use a liquid scintillation counter to determine the 35S counts in each tube. labelled Eppendorf tubes with 25 µl of deionized formamide (this will be the final storage tube for the purified 35S-cRNA). as follows (this is a typical in vitro transcription result): You purchased 1 mCi of 35S-UTP (concentration. Add 4 ml of aqueous-miscible scintillation cocktail to scintillation vials A and B. Remove 1 µl of this purified probe from each tube. so that the entire reaction contained 298 pmol of (hot + cold) UTP -. and you used 4 µl or 48 pmol of 35S-UTP/reaction. The 12.0 pmol/µl of isotope. Microfuge the tubes for 3 . Vortex and pulse-spin the tubes for 4 counts. Resuspend the pellet in 25 µl of DEPC-H2O (this time notice that the pellet can be resuspended very easily)..e. During this incubation. the 1 µl of nucleotide cocktail for your in vitro transcription reaction contained 250 pmol of non-radioactive UTP. probe) volume of ≈50 µl.≈6. 10. not just the UTP content.5 mCi/ml of the purchased product equates to 12. CTP & GTP). It is critical to avoid aspirating any glassmilk at this point. leaving you with RNAse contamination of your purified preparation). Transfer this eluted cRNA into the deionized formamide-Eppendorf tubes (step 8).2 times the amount of UTP you would detect if you were measuring radioactivity alone. so remove the supernatant 10 µl at a time using your 1 .10 µl pipetter. . and dot it onto a filter paper disc (labelled B).2 x 4 9.. set up some new. Transfer the purified probe to a -70˚C freezer until you are ready to use it. Calculate the reaction yields (i. Since we are interested in calculating the amount of cRNA synthesized. Repeat step 7 for a total of three washes. inside the tip. as above. 1000 Ci/mmol = 1000 mCi/µmol) in a 80 µl volume. ng cRNA synthesized and purified) for each probe generated.5 min (full speed) to pellet the 'glass milk' and carefully remove the 35S-cRNA (supernatant)) from each tube. Transfer the paper disc into another scintillation vial (labelled B) for ßcounting. In addition. so that the actual amount of cRNA synthesized will be approximately 6. so that you should now have a total 35ScRNA (i. will 'bump' the solution up onto the end of the micropipetter.77 8. we must also take into account that the mass of cRNA synthesized contains all four nucleotides (UTP.
(pmol 35S x 24. into cRNA (x106) 28.2 21. into the cRNA probe 24. we can probe between 21 and 26 slides for each of the cRNAs.) 26.78 = 24. for these in vitro transcription reactions. (pmol/2) No.8) 605. slides (11.8 38.5 incorp.3 (cpm/pmol) x106 TGFα-s TNF-s TNFα-s 5.2 ng cRNA synth. Now. The raw data obtained from the 1 µl aliquots taken in steps 3 & 10 is: probe TGFαs TNFs TNFαs step 3 (cpm) 569150 823920 596450 step 10 (cpm) 576280 768370 494110 specific total cpm probe per rx.3 24.9 278 245.4 302. we will determine the amounts of cRNA synthesized.18 1. the total cpm put into the reaction & the total cpm actually incorporated into the cRNA.72 1.5 ng cRNA ea.4 19.4 22.24 5.5 24.69 8.8 times the levels of 35S-UTP incorporated (this calculation assumes that UTP comprises 25% of the total nucleotide content). (x107) activity of 35S pmol 35S total cpm incorp. respectively).75 pmol cRNA synth.24 Thus.8 556 490. . using the cpm measured in the 1 µl aliquots taken from the transcription reactions (steps 3 & 10.96 1.
Since cytocentrifuge preparations are not embedded in paraffin and are stored already in 70% ethanol. Reagents 10 M NaOH xylene ethanol (100%.2 Preparation of slides for hybridization Materials 37˙C water bath 60˙C water bath 90˙C water bath slide-staining apparatus (12 'coplin' jars are adequate) 10 ml glass pipettes (baked or commercial disposable) micropipetters (P200 & P2000) and tips 50 ml graduate centrifuge tubes (adequate for measuring reagents) pH paper strips (0. when you are doing ISH with paraffin sections.5 min .40 µg/ml in TE. then adjust pH to neutral). Normally.2.1 M triethanolamine (prepare just before use) acetic anhydride METHOD 1.2 M HCl (16 ml concentrated HCl in 984 ml DEPC-H2O) TE buffer (10mM Tris/1 mM EDTA in H2O) PBS proteinase K (1 .79 4.5 pH unit sensitivity is fine). and then are treated as with the paraffin sections protocol. they enter the process at the 70% ethanol stage. 0. 90%. you need to first de-wax and rehydrate the sections with sequential treatments with xylene and ethanol. Therefore process your slides as appropriate by running them through the following baths (in order and for the specified times): xylene #1. & 50%) DEPC-treated H2O (≈500 ml) 0. 0.10 min xylene #2. from frozen stocks). 70%.2% glycine in PBS (3 ml of 10% glycine IN 147 ml PBS) 4% paraformaldehyde in PBS (dissolve at 60˙C at high pH. prepare immediately before use.
70%.40 µl aliquots and drop these onto the paper). During the 20 min 0. While the tissues are in the paraformaldehyde. Rinse the paraformaldehyde out of the cells/tissues by immersing in PBS for 5 min. 3.8) by adding ≈600 µl of 1 M HCl. and then neutralize the pH (to ≈7 .. To prepare the 0.8.71g triethanolamine powder to 200 ml DEPC-H2O. after the TE equilibration (step 4). The paraformaldehyde will not dissolve until the PBS is made basic (i. For cytocentrifuge cells it is often acceptable to use the lower concentration of proteinase K. 4. transfer the slides into a TE bath for 5 min 5. increase the pH) by adding ≈6 drops of 10 M NaOH. Thus.40 µg/ml).2 M HCl incubation period (i. while for paraffin sections you should use the highest concentration that does not damage the integrity of the tissue (often 25 . Block the non-specific binding sites of the 35S-labelled probes by incubating the cells/tissues in 0. prepare a fresh solution of 4% paraformaldehyde by adding 6 g of paraformaldehyde to the 150 ml of preheated 60˙C PBS. 90%. After the 20 min HCl hydrolysis step. ≥30 seconds each).2% glycine in PBS for 2 min 7.(4 baths total.e. transfer slides into the proteinase K/TE bath for 15 min @ 37˙C. 2. use pH paper. & 30% ethanol . Hydrolyze the overly extensive protein cross-linking in the formaldehyde-fixed tissues by incubating them in 0. not a pH meter).2 M HCl for 20 min (this increases the access of your ISH probes to the mRNA). move it to an ice bath to cool. 8.5% acetic anhydride for 10 min. 10. It is critical that the complete triethanolamine/acetic anhydride blocking solution is . Neutralize the newly-exposed RNAses in the tissues by "post-fixing" them in the freshly-prepared 4% paraformaldehyde in PBS (from step 3) for 5 . 9. again. step 2). Further break down the protein-protein cross-linking (to increase access of the probe to the mRNA) by digesting the cells/tissues with the highest levels of proteinase K that they can take without disintegrating due to over digestion. Equilibrate the cells/tissues to PBS for 3 min.80 100%. Neutralize the progression of the proteinase K tissue digestion by immersing the slides in 0.1M triethanolamine containing 0. Swirl the basic the paraformaldehyde suspension until all of the paraformaldehyde is dissolved. and then adjust the pH to 7. 6.5 .1 M triethanolamine. add 3. When the paraformaldehyde is at the correct pH. prepare the triethanolamine solution for the tissue blocking step below.20 min on ice.. but instead remove 20 . Check the pH of the solution with pH paper (do not dip the paper in the solution.e.0 by adding 200 µl of 10M NaOH (additional NaOH may be needed.
briefly remove the slides. below)..e. add another 500 µl of acetic anhydride (acetic anhydride is extremely labile and 'goes off' within minutes). during the last few seconds of the step 9 PBS rinse. and continue the blocking step for another 5 min (i.e.e. total blocking time.81 prepared fresh immediately (i. hybridization step.. Transfer the slides to a 2xSSC bath and hold in this solution until you are ready for the probe application to the tissues (i. Therefore.. 10') 11. seconds) before use. . After 5 min. add 1 ml acetic anhydride to the 150 ml bath of 0.1M triethanolamine and immediately immerse the slides in this mixture.
5 pH unit sensitivity is fine).2. for additional blocking of non-specific binding of 35S-UTP) 35S-UTP-labelled sense and anti-sense cRNA probes (concentrations known) METHOD 1.82 4. Calculate the amounts of probe. thio-UTP (1. buffer (tot-thio-probe) 1087. Reagents DEPC-treated H2O (≈500 ml) ISH hybridization buffer thio-UTP (non-radioactive. and applying 45 µl of final hybridization cocktail to each slide.5 30 27 Volume cRNA probe* (11. hyb. (45µl ea. thio-UTP and hybridization buffer that you will require for your experiment. -the 45 µl for each slide must include 1.0 kb = 11.25 ng x 1. Base these calculations on: -using a final probe concentration of ≈0.25 ng/slide.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA Materials 40 .25 µl/slide) 32.5 1000 868 .25 µl of thio-UTP -the balance of the 45 µl for each slide comprises hybridization buffer A typical set of calculations for a ISH procedure in which you are going to probe 8 sense (negative control) slides and 20 anti-sense (experimental) slides with 35S-TNF cRNA probes that have cRNA concentrations of 5 ng/µl is as follows: cRNA TGF: anti-sense TNF: sense TNF: anti-sense # slides 26 24 21 Tot.65˙C hybridization oven 90˙C water bath micropipetter (P200) and tips pH paper strips (0. then you will need 45 x 0.) 1170 1080 945 Vol.5 ng/slide) 50 50 50 Volume hyb. vol. For a 1 kilobase cRNA probe.25 ng of probe/µl of final hybridization cocktail/kilobase of cRNA probe complexity.
A few minutes prior to using each probe. 5. . remove the slides to be probed from the 2xSSC bath. carefully using the pipette tip to cover most of each tissue section (for cytocentrifuge preps simply placing the 45 µl in the centre of the cell 'patch' is adequate). Add each required reagent to labelled Eppendorf tubes. 4. and place on hybridization tray (over 50% formamide-2xSSC-soaked paper towels in a Tupperware container). Apply 45 µl of the final hybridization mixture to each slide. Use some RNAse-free forceps to carefully overlay the tissue/cells with a baked. when finished. then transfer it back onto ice to quench the renaturation process. Kleenex). taking care to avoid air bubbles. place it in a 80 90˙C water bath for 2 min to denature the cRNA. siliconized coverslip.. cover and seal the hybridization chamber (Tupperware container) and place it in the 45˚C hybridization oven overnight. vortex briefly and hold on ice until ready to use. Repeat this procedure in turn with each of the slides and.g. 3.83 2. One at a time. briefly dry the back and the sides of the slides with a clean Kim-wipe (e.
by incubating the slides for 30 min at 37˙C in RNAse (20 µg/ml) in 4xSSC-TE. Digest the single-stranded 35S-cRNA that is non-specifically bound to the tissues (i. 70. away from the radioactive area). Equilibrate slides for 15 min at 37˙C in 4X SSC-TE [4X SSC containing 10mM Tris and 1 mM EDTA] 4.. & 90%) each containing 0. for a total time of 60 min. Discard the 2xSSC solution from step 1. Discard the used RNAse digestion buffer in the liquid 35S waste bucket. 2. Wash the residual RNAse from the slides for 30 min at 37˙C in 4xSSC-TE . not hybridized to mRNA) with RNAse A. Remove each of the slides from the hybridization chamber and place.e. 5. 3..2.e.84 4. handle the slides by their frosted ends. 30.4 Post-hybridization washing & autoradiography Materials 37˙C water bath 47˙C water bath 2 coplin jars bath for removing cover slips Geiger counter plexi-glass shield for working with 35S isotope disposal bins Reagents 2x SSC 50% formamide/2xSSC/10 mM 2-mercaptoethanol 4xSSC/TE RNAse A (10 mg/ml) ethanol (i. Transfer slides to a tissue-tek holder and incubate for 30 min at 50˚C in 50% formamide-2xSSC-10mM 2-mercaptoethanol. 50. as well as the used contents of the two reagent baths from step 2 in the liquid 35S waste bucket.3 M ammonium acetate 100% ethanol METHOD 1. in the metal slide holder (as much as possible. Immerse the slides in a bath of 2xSSC until each of the coverslips has fallen off of the slides. After 30 min repeat this step. back-to-back.
Subsequent steps are performed in the darkroom under safelight illumination: 10. Return the unused emulsion from the mailer tube to the centrifuge tube and return it to the refrigerator. 13. Dip a clean (blank) slide in the emulsion and check for smooth. 8. fill vertical slide mailer with emulsion. Return the slides to tissue-tek holder(s). scan each with a Geiger counter to get a feeling for the levels of radioactivity associated with each (this will determine roughly the exposure times for the autoradiography -. gently dip each experimental slide in the emulsion. place each into black slide boxes. each containing 0. wrap in 2 layers of foil. immediately adjacent to the painted or frosted surface of the slide). Complete the dehydration process in a 100% ethanol bath (30 sec) and then transfer the slides to a paper towel on the bench and allow them to air dry for 5 -10 min. remember clearly exactly how you have allocated your slides. 12.3M ammonium acetate). One at a time. 7. When the slides are dry. Discard the used wash solutions in the liquid 35S waste bucket. keeping the slides in each for ≈30 sec. Also prewarm a the slide mailer. withdraw it and wipe the back with a glass slide or razor blade and then standing vertically against the inner sides of drying boxes.85 6. and store in a -20˚C freezer. digested 35S-UTP (or larger nucleotides) by incubating the slides for 60 min at 50˚C in 50% formamide-2X SSC-10mM 2-mercaptoethanol. Place the covers on the boxes so that they are light-proof (which will allow you to open the door of the darkroom and walk out) and allow slides to dry for 30 min. Perform one final high stringency wash to remove non-specifically bound. 9.e. 11. 50%. label with tape. Allow 10-15 min for the emulsion to melt. on a clear section of the glass that will not be come in contact with the autoradiography emulsion (i. With the slides laid out flat on paper towels. When the emulsion is melted. clearly separated into "early" and "late" development groups. and label each of them with some sort of identification number.short or long exposure time). and 93%. under safelight illumination. Place an aliquot of emulsion in alight-proof holder (half-filled with water) within a 42-45˚C water bath. so they are best done with a magic marker. Since all subsequent steps are performed in the darkroom under safelight illumination. "bubble-free" coating. 70%. These numbers will need to be visible in the darkroom. and then dehydrate them by sequentially transferring them through a graded series of ethanol baths (30%.. Equilibrate the slides to 2xSSC by incubating them for 3-5 min (at room temperature) in this solution. .
then to xylene #1 for 2.2% toluidine blue in 60% ethanol bath acetone bath (2) xylene bath (2) entellen or permount mounting medium METHOD 1. Transfer slides into 60% ethanol for ≥30 sec. followed by a second acetone bath for another 2.5 min. Transfer to xylene #2 for 2.2% toluidine blue in 60% ethanol for ≈30 sec Dip the slides in the water bath 3 times and then transfer to the first acetone bath for 2. In the darkroom.5 min (complete submersion is required to remove all H2O.5 min. and finally in the fixer for 5 min.5 min. transfer the slides to a tissue tek slide holder.5 Autoradiograph development & counter-staining Materials staining coplin jars slide staining rack cover slips Reagents Kodak D19 developer (diluted 1:1 with H2O) H2O Kodak rapid fixer running water bath 60% ethanol bath 0. it is safe to turn on the lights). avoiding air bubbles. which will irreversibly turn the dehydrated emulsion opaque) 3.2. and then place them first in the D19 developer for 2. 4. Soak the slides in gently running water for ≥2 hours. 7. . 2. then into the 0. (Within a minute or two of transferring the slides into the fixer. under safelight illumination. 6.86 4. 5. Remove slides to be developed from the freezer and allow the still foil-wrapped boxes to come to room temperature.5 min. then in the H2O for 15 sec. Place a drop of permount on the slides and coverslip carefully.
1) oligo(dT) DEPC-treated H2O 10x PCR buffer () 50 mM MgCl2 10 mM dNTP mix (dATP. Materials thermocycler 42 & 70˙C water baths & an ice bath micropipetter (0. actin -many propose that the levels of actin mRNA change little as a result of cellular stimulation). PCR primers for most of the human and mouse cytokines are readily available commercially. Protocols specific for individual cytokines (see §5.1..5. e. then comparing the signals of each reaction after the PCR amplification. dGTP & dTTP) 100 mM dithiothreitol (DTT) M-MLV RT (murine Moloney Leukemia Virus reverse transcriptase. No. either as pre-packaged or as custom-synthesized products. dCTP. Appendix E: Human cytokine RT-PCR primers) are usually published with the sequences in the literature. 200 U/ µl) 3' & 5' oligonucleotide primers (target mRNA-specific.g. and using these will often circumvent your having to develop your own protocol. actin & cytokine primers) Taq DNA polymerase .5-10µl) and tips horizontal gel electrophoresis apparatus and powerpac Reagents purified total cellular RNA (see §4. The ratios of the cytokine signal to that of actin will change if the mRNA levels for the cytokine were altered as a result the original cell or tissue stimulus or pathophysiologic process(es). as well as that of a house-keeping gene of interest (e. The method is made semi-quantitative by virtue of amplifying cDNA for both the cytokine of interest. The protocol proposed herein is a generic one which is based on the GIBCO/BRL product instruction manual for their PCR kit (Cat. 18089-011).g.87 4. The sequences for custom synthesis are obtained from the scientific literature..3 Semi-quantitative RT-PCR to detect cytokine mRNA Polymerase chain reaction (PCR) amplification of cDNA from reverse-transcribed mRNA is perhaps the most powerful method of detecting transcripts in cells.
6. then mix and spin briefly. 3. and collect by brief centrifugation. Incubate each sample at 70˚C for 10 min to denature the mRNA and then incubate on ice for at least 1 min. adding each component in the indicated order (for n samples -.5 ml tubes as follows: 1 to 5µg total RNA x µl oligo(dT) 1 µl DEPC water to 12 µl (total volume). 7. Prepare RNA/primer mixtures in sterile 0.88 agarose TAE buffer ( mM Tris/ mM sodium acetate/ mM EDTA. Add 1 µl (200 units) M-MLV RT to each tube. Incubate at 42˚C for 5 min. pH ) ethidium bromide (10 mg/ml) DNA sample prep buffer 100 bp DNA oligonucleotide ladder (molecular weight standards) 4. mix gently. Terminate the reactions at 70˚C for 15 min.) Component Each reaction (µl) 10X PCR buffer 2 50 mM MgCl2 1 10mM dNTP mix 1 0. Add 7 µl of reaction mixture to each RNA/primer mixture. mix and incubate at 42˚C for 50 min.3. 5.1 First strand cDNA Synthesis using Oligo(dT) priming This procedure is designed to convert 1 to 5µg of total RNA into first strand cDNA 1. . The samples are now ready for PCR amplification reaction. Chill the tubes on ice. Prepare the following reaction mixture. 2.1M DTT 2 DEPC H2O 1 4.but prepare the reaction mix for n+1 reactions to ensure sufficient levels for all reactions.
programmed as follows: 1 cycle 94˚C 3 min (initial denaturation) 30 cycles: 94˚C 30 sec (cycle internal denaturation) 55˚C 30 sec (primer-target annealing temp) 72˚C 1 min (primer extension rx) hold at 72˚C for 7 min to complete the last extension. and mix well.5 x n µl 3'-primer 0.5 x n µl Taq DNA polymerase for each reaction. 1. add 2 µl RT reaction product to 48 µl of above mixture (use 0. Place the samples in the thermocycler.89 4. 3. 4. . Prepare the following reaction mixture (for n samples). 25 x n µl 2x stock buffer 0.5 x n µl 5'-primer 21.5 x n µl H2O 0. Make the 2x stock buffer (recipe for 500µl): 100µl 10x PCR buffer 30µl 50 mM MgCl2 20µl 10mM dNTP mix 350µl DEPC H2O 2.5ml tubes) and mix well. Analyze 5-20 µl of the amplified sample by agarose gel electrophoresis.3. Use only 10% of the first strand reaction for PCR. Adding larger amounts of the first strand reaction may actually decrease the amount of product synthesized. then hold indefinitely at 4˚C until ready to analyse.2 PCR amplification of the target cDNA The first cDNA may be amplified directly using PCR.
3 µl Heat the water/agarose to boiling in a microwave to dissolve the agarose. and examine the banding patterns and relative amounts of PCR products in each lane using computer-assisted image analysis of the illuminated gel (or alternately by eye). so do not pour the gel into the mold until it is sufficiently cool .3.26 g 40x TAE buffer 0. Prepare the actin and companion cytokine PCR reaction products and 100 bp molecular size standards for gel analysis by heating to 65˚C for 10 min in DNA sample prep buffer containing 1µl ethidium bromide. 4. 5-20 µl of the amplified sample 5-10 µl DNA sample prep buffer 1 µl ethidium bromide Load the samples and 100 bp ladder into the wells and run at 50-70 mA until the lead dye front has migrated approximately 2/3 of the way through the gel. with sufficient lanes to run each of the samples and a 100 bp DNA oligonucleotide ladder. Place the gel on an ultraviolet light-box.3 ml agarose 0. (Be sure to allow the gel to polymerize sufficiently such that the bottoms of the well do not break when removing the well-forming comb. 20 At temperatures above 56˚C the gel apparatus will warp badly. approximately 7 mm thick. 4. 3.2 Detection of RT-PCR products Pour a 1. then cool this solution to 56˚C and add the TAE and ethidium bromide20.90 1.825 ml 10 mg/ml ethidium bromide 3. 2. or accurate band comparisons will not be possible.) water 32. Be sure that the individual band signals have not reached the saturation point for the image analyser.2% agarose-TAE buffer gel.
GENERAL METHODS 5. 5. Titrate the serum for SRBC agglutination activity.g.91 APPENDICES: 5.. Transfer ≈10 . top and bottom ones). Capillary activity will draw the cells under the cover slip and thereby fill the viewing chamber 5. first at low power setting and when you have your bearings under the scope. Pipette mixture up and down several times to mix the cells 4. . Two weeks later inject them again with 200 µl of a 10% SRBC suspension. Inactivate the complement (C') cascade activity of the serum by heating it to 56C for 60'. and allow it to clot in a glass tube.2 Cell counting with a hemocytometer Materials hemocytometer & coverslip single cell suspension of cells to be counted 20 µl micropipetter and tips eppendorf tubes 0.20 µl of the cell suspension to the hemocytometer (with coverslip in place). 5. 2. central . Count the total numbers of cells in each of 5 of the larger squares (e..1. Examine the cells under the microscope. I always include those on the top and left-hand side lines).4% trypan blue in saline METHOD 1. Mouse anti-SRBC antiserum can be prepared as follows: Inject BALB/c mice with 200 µl of a10% suspension of SRBC in PBS. Cap and gently invert or otherwise swirl cells several times to ensure an even cellular distribution 2.1 Anti-sheep RBC antisera (preparation of) 1.g. right. Withdraw 20 ul of cells to a 0. You will note that the chamber is divided up into 9 larger squares. You should count all cells that lie inside the boundary lines of the squares.1 APPENDIX A -. e. pick whichever ones are easiest for you to remember.5 ml eppendorf tube and add an equal volume of 0. first for 1 h at room temperature and then overnight at 4˙C. 4. noting the cell count in each. switch to higher magnification for actual cell counting. each of which is in turn subdivided.5 ml push button counter microscope Reagents 0. left. 3. Approximately one week later collect blood from the mice. 6. and you should also count the cells that fall on top of the lines delineating two of the four sides (for consistency.4% trypan blue 3.1.
use the following formula: viability (% ) = 10. To coat the zymosan beads with C3b. then you might simply vigorously pipette the cells in the eppendorf tube (i. For example. if you had a mean of 24 cells/large square.B. you have to decide whether you can get an accurate estimate of the cell numbers with the clumps present. . the trypan blue/cell mixture) to disperse the clumped cells and then recount.1. add 200 µl of fresh mouse serum to 2 mg 'activated' zymosan and incubate for 15 min at 37˙C. However. 2..92 HEMOCYTOMETER FIELD square 1 2 3 4 5 mean cell count 4 3 1 2 4 2. If your cell preparation includes clumps of cells. 3.8 7. if the cells are very badly clumped. using H2O followed by 70% ethanol. Wash the yeast with PBS and store at 4˙C until ready to use. In addition. 5.1 mm3 (or 10-4 cm3). 9.e. since you used equal volumes of cell suspension and trypan blue for counting. Each of these squares contains total volume of 0. you will have to disperse the cells in the stock suspension. and allow to air dry. either by vigorous pipetting (which is very inefficient) or by re-centrifuging and dispersing the cell pellet correctly. but not healthy cells). to calculate the cell concentration (in cells/ml) in your original cell suspension. take the mean numbers of cells in the large squares and multiply by 2 (your dilution factor) and by 104 (volume adjustment). you have diluted your cells two-fold. N. Determine the numbers of non-viable cells by counting the numbers of cells with nuclei that are stained intensely blue (trypan blue is a vital stain for dying. If you feel that the clumps are evenly distributed in your original cell suspension. Activate the zymosan A by boiling for 10 min in PBS and then washing with PBS. Resuspend to a final concentration of 10 mg/ml.3 C3b opsinization of yeast (zymosan A) 1. ⎛ # unstained cells ⎞ × 100 ⎝ total # stained + unstained cells ⎠ Clean your hemocytometer immediately after use. 8. Calculate the mean number of cells (both viable and non-viable) in each of the 5 larger squares. the concentration of your original cell suspension would be: 24 x 2 x 104 = 48 x 104 cells/ml To calculate the cellular viability in your preparation. Therefore.
5 M stock/996. add 50 . prepare cell suspension of ≈5x105 . assemble centrifuge chambers with labeled slides and filters in correct orientation and load chambers into the centrifuge. filters. finally. concentrations. etc.for most purposes.) 4. Materials hot plate beakers dialysis tubing of required size (and molecular weight cut-off) Reagents 0..105 cells/slide. then transfer the tissues/cells to 70% ethanol and store at -20˚C until ready to process to paraffin blocks by routine methods.05% EDTA 3.106 cells/ml 2. 4.05% Na2CO3 (0. centrifuge the cells for 4 . cut tubing to suitable sizes. you want to have somewhere in the order of 5x104 . when the rotor stops. boil a third time for 10' in 0. . stored either at room temperature or in the freezer. Fix 3-6 mm blocks of tissue for 3 h (or cell suspensions for ≈30 min) on ice in ISH fix (§5. clean the disassembled chambers and clamp assemblies with H2O and allow to air dry after use.93 5. Ideally. usually in an area about 6 -8 mm in diameter. Dialysis tubing (preparation) Dialysis tubing can be prepared well in advance and stored in the refrigerator for extended periods of time. clamps.6 Fixation of tissues for ISH or IHC.) glass microscope slides METHOD 1.. rinse several times with H2O and. 3. Materials cytocentrifuge (with chambers.. remove slides from the chambers (being careful not to scrape the cells off of the slides in this process) and allow the cells to air dry (alternately. you can fix the cells in acetone or alcohol for ≈15 sec and then air dry) 6.1. or 3.200 µl of cells to each chamber (depending on the cell numbers. Most dialysis tubing has been treated with glycerol by the manufacturer in order to prevent excessive drying out (and cracking) during storage -. 7.4 Cytocentrifuge preparations The cytocentrifuge is simply a centrifuge that deposits cells from a suspension culture directly onto a microscope slide.05% NaCO3. this glycerol should be removed from the tubing before use.1. depending on their purpose. allowing for tying or clamping ends or prepare large sections 2..5 g/l.3).5 g/l H2O) distilled H2O 50%ETOH METHOD 1. boil the sections of tubing for ≈10' in 0. etc. the air-dried cells can be stained immediately or.5 min at ≈1500 rpm 5. boil a second time for 10' in 0. It is sometimes to convenient to prepare a very large batch so that you will have it on hand whenever you need it. It is an ideal way to prepare the cells for microscopic examination.6 ml H2O) 0.05% Na2CO3.05% EDTA (0.4 ml of 0. 5. store at 4˙C in 50% ethanol (keep the beaker covered with parafilm) 5.
you can lyse them by incubation in ammonium chloride lysis solution for 5 min 5.. needle on a10 ml syringe. 5. 5. in MEM medium. 2. fractionating the cells by density gradient centrifugation. The lungs of animals undergoing strong pulmonary challenges with allergen or other disease agents often contain very high numbers of perivascular and peribronchial lymphoid cells.. and wash the dispersed cells in DMEM-10%. add 1 volume of 10x HBSS to the H2O suspension of cells and rapidly disperse it throughout the cell suspension by swirling or inverting the (capped) tube.1. Alternately.5 mm3) with a scalpel. add 9 volumes of double distilled H2O to the cells and vortex the tube by hand to rapidly disperse the cells throughout the H2O. Briskly flick the tube to resuspend the cell pellet (to an even paste on the walls of the tube). MEM density gradient media (e. .g. anaesthetic. Filter the digested tissues through ≈4 layers of sterile gauze to remove undigested tissue fragments.8 Lysis of red blood cells There are a number of options available for lysing contaminating RBC's in a cell preparation. if necessary. which takes advantage of the fact that red cells undergo lysis in H2O very quickly.B. The immunologic reactivity of the lung tissues can be profoundly different than that of the spleen or other non-pulmonary lymphoid organs.1. 3. including commercially available preparations. When the clock reaches 0 seconds. 6. carry on with the purification. surgical instruments. Obtain lung tissues and dice them finely (to ≤0. Percoll. 2. ideally on a rocker platform. Determine the cell yield and viability by direct counting of trypan blue-treated cells using a hemocytometer. Disperse any undigested fragments of tissue by repeated aspirating through a 20 ga. 15 ml polypropylene tubes Reagents DMEM-10%. Wash the cells two times in DMEM-0% FCS to get rid of the RBC ghosts (plasma membranes).8. when it reaches 15 seconds. and aspirate all of the medium from the cell pellet.1. optional) collagenase (Worthington Scientific). and in some species these animals also develop discreet collections of BALT. 3. a very brief pulse with H2O will lyse all of the red cells and leave the WBC intact.. Therefore. The signals from both ISH and IHC procedures seem to be superior if the tissues are processed to paraffin expeditiously rather than holding them for prolonged periods in 70% ethanol.1 Hypotonic lysis with H2O 1. 5. Either use the cells directly or.7 Lung cells (single cell suspension) Single cell suspensions of lung cells are very useful for the determination of immunologic reactivity of the lung associated immune compartment. TIMING IS CRITICAL! 5. hemocytometer CO2 incubator. while nucleated cells are damaged much more slowly.75 mg/ml hyaluronidase METHOD 1.5 mg/ml collagenase and 0. Start a countdown timer set to 25 or 30 seconds and. Materials mice.94 N. hyaluronidase (Worthington Scientific) MEM containing 1. One of the most simple is hypotonic lysis. Lymphocyte Separation Medium. Sediment all of the cells in your preparation by centrifugation. 4. Transfer the tissues into fresh MEM containing collagenase/hyaluronidase (≈1 g tissue/10 ml enzyme cocktail) and incubate at 37˚C for 60 min. 4..
Wash the cells with medium and store at 4˙C until ready to use. 3. Generate a 0. and 100 µg/ml.1% SRBC. and bleeding it ≈3 wk later).try standards of 5. 5. which is a good long-term storage reagent for SRBC.1. 25.11 Splenocytes (single cell suspensions) Materials BALB/c mouse anaesthetic (methoxyfluorane) clinical centrifuge. Measure the absorbance at a wavelength of 595 nm.1. Add 40 µl of the diluted dye reagent to each well and mix the samples thoroughly by repeated pipetting. Resuspend the cells in 5 ml of ammonium chloride lysis solution and let stand for 5 min. Perform steps 1 & 2 as above in the hypotonic lysis protocol 2. (anti-SRBC antiserum can be generated by vaccinating a mouse with 0. 2. 1 part reagent + 4 parts H2O). 15 ml centrifuge tubes (polypropylene [pp]. (store at 4˙C. 5. and incubate for 30 min at room temperature.1. and filter through Whatman #1 filter paper. 3.10 Protein assay in microtiter plates Materials pipettes. dilute BSA standards to a range that should bracket that of the unknowns -. stable for 2 weeks) 2. dilute dye reagent 1:5 with H2O (ie. . tips. Pipette 160 µl of standard and sample solution into separate wells of the microtitre plate 4.2 ml of 0. Wash the cells two times in DMEM-0% FCS. but no more than 1 h.2 Lysis with ammonium chloride 1.5% suspension (vol/vol) of sheep red blood cells21 (SRBC) in PBS. The cells are washed two times with Alsevers solution (see Appendix C) and resuspended in Alsevers solution. add 50 µl of heat-inactivated mouse anti-SRBC serum to 300 µl of the SRBC suspension. as above 5. To coat the cells with anti-SRBC.8.9 Opsinization of SRBC with antibody 1. 3. multichannel pipetter 96 well plate microplate reader filter paper Whatman #1 Reagents: Bio-Rad concentrated dye reagent protein standard (we will use bovine serum albumin.95 5.0. BSA) METHOD: 1. 50. 10. not polystyrene [ps]) pipettes/pipettors (micropipettes and macropipettes) Reagents DMEM/10% FCS (Appendix B) 70% ethanol optional: 21 SRBC are obtained by venipuncture (usually the jugular vein) of sheep directly into EDTAcontaining syringes or alternately into regular syringes followed directly by transfer of the blood into EDTA-containing tubes. Incubate the plates at room temperature for at least 5 min. 5.4.
examine the cultures to confirm that the cells have aggregated as they should following ConA stimulation. Holding the skin over the spleen up into a tent. 2. Place the spleen in a petri dish containing DMEM-0%FCS and tease the tissues apart using two pairs of fine. etc. or DMEM or RPMI. 3.25 times. When the mouse ceases breathing (very shortly after step 1). Aliquot the supernatants and store at either -20˙C or -80˙C. This conditioned medium will keep its activity for many months.4 layers of sterile gauze drawn across the top of a 15 ml centrifuge tube. Filter the dispersed spleen cell preparation through 3 . Pull the skin open and reflect it full back.g. this will usually be 3x106 cells/ml of DMEM-10% FCS. pull the mouse backwards by the tail. Add ConA to the cultures to a final concentration of 4 µg/ml and place the cells in the CO2 incubator for 4 days. You will hear a popping sound as the neck dislocates.) METHOD 1. vigorously pipette the cells 20 . firmly place an instrument (e. To dislocate the cervical spine effectively. Wash the cells once by centrifuging and resuspending to the desired final concentration in the desired medium. Open the body wall over the spleen with the scissors. firmly.12 Splenocytes (spleen cell-conditioned medium) Materials single cell suspension of spleen cells clinical centrifuge.12x106 nucleated cells. and soak the left side with 70% ethanol. 15 ml centrifuge tubes (polypropylene [pp]. 5. forceps or closed scissors) across the back of the neck and holding the mouse in position with this instrument. Prior to harvesting the cells. 4. forceps) METHOD: 1. both dorsally and ventrally. 3. lay it on its right side. you may obtain anywhere from 2 . . For our purposes. pull the spleen up from the other viscera and clip away the vascular attachments and fat. 2.96 sterile surgical tools (scissors.. Using a pipette aid (electrical pipetting device) and a 10 ml pipette. Set up the cells in a T75 flask. Continue teasing until all of the tissue clumps are dispersed as much as possible. Generate a single cell suspension of spleen cells from a normal mouse with a cell concentration of 3x106 cells/ml of DMEM-10% FCS. 5. and then grasp both sides of the incision firmly between the thumb and forefinger of each hand.1.g. to completely break up the clumps. From one normal mouse spleen.. place the mouse down on the bench in sternal recumbancy (belly down). Remove 20 µl of the filtrate (now a single cell suspension) for hemocytometer counting. curved forceps. 4.. not polystyrene [ps]) pipettes/pipettors (micropipettes and macropipettes) T75 tissue culture flasks Reagents: DMEM/10% FCS (Appendix B) concanavalin A (4 mg/ml stock solution in PBS. Euthanize a mouse (e. but not too forcefully. transfer them to 50 ml centrifuge tubes and sediment the cells by centrifugation for 10 min at 1500 rpm. Provided the cells appear as they should. incise it with a pair of scissors. BALB/c) by inducing surgical-level anaesthesia with methoxyfluorane and dislocating the cervical spinal column.
Apply 50 . Most circulating lymphocytes are unstimulated ones and appear as very small cells that contain large nuclei. The cells can be differentiated based on their staining and morphology. & 50%) isopropanol METHOD 1. In fact. with few if any indentations. In effect: POLYMORPHONUCLEAR CELLS (all have highly lobulated nuclei) --eosinophils have rather large. 5.13. rinse the slides with water as in step 3 and briefly look at the sections under the microscope.1 Wrights-Giemsa staining and morphologic identification of PBL Materials cytocentrifuge cell preparations. incubate for 1 min.13.1 Giemsa stains 5. red-stained cytoplasmic granules that fill all of the extranuclear compartment of the cells. After one hour. often the cytoplasm appears as a small rim of powder blue-coloured cytoplasm. 2x 30" 100% ethanol. flush the scum from the slide with ≈0. 4.100x power using a compound microscope.1. 1x 30" 95% ethanol. but they will probably serve to fulfill most of your needs as far as differentiating these cells. 1x 30" 50% ethanol..1.100 µl of stain to the cells on each slide. and allow to sit for 10 min.13 Staining Protocols 5.lymphocytes are present in substantial numbers in a number of compartments. with nuclei the same size as that of the small lymphocytes. Examine the cells under 40x . --in the mouse.1. then air dry standing up. Transfer the slides into the Giemsa stain bath & hold for 1 . 70. Results The appearances of the different types of mouse PBL following Giemsa staining are demonstrated below.5 ml of buffered water 3. Wrights-Giemsa stain (10% Wrights-Giemsa solution in buffer) neutral buffered water METHOD 1. as in figure x. monocytes are much like large lymphocytes. 1x 30" 70% ethanol.97 5. 100% ethanol (& 95. Activated lymphocytes (plasma cells) tend to be large. basophils are present in such low numbers that some authors state that mice do not have basophils.2 h . but they have abundant cytoplasm. 2. MONONUCLEAR CELLS (all have round to slightly indented nuclei) -. Mount coverslips on the slides with Permount or Entellen 5.13. Add 100 µl of neutral buffered water to the cells.1. They appear much like monocytes. Deparaffinize and rehydrate the tissue sections (2x 5' in xylene.2 Giemsa staining of tissue sections Materials tissue sections on slides Giemsa stain xylene. You probably will never see a mouse basophils (unless you begin working with these cells. These are very simplified descriptions of the WBC. -. The nuclei are usually very round. If they .neutrophils have rather small. etc. with one or two mediumsized deep purple-staining granules.1. but the nuclei are usually indented. 2.1. pale pink-stained cytoplasmic granules that fill the extranuclear compartment of the cells.
13. Transfer the slides to 0. and finally two changes of xylene (also 2-3 min each).2 Gills hematoxylin for IHC Materials Gill's hematoxylin solution (1:5 dilution of commercial stain in H2O) 10% methanol. Cover-slip the slides with aqueous mounting medium. proceed with step 4. add 1 µl of stock cytokine (in this case. 1. 4. 0.5 min). Mount coverslips on the slides with Permount or Entellen 5.04. Transfer the slides into the TBS for ≈1 min (to blue or differentiate the stain). 4.5 min each).2% toluidine blue in 60% ethanol and hold for 30 seconds. 3. such that you are .004 U/well). H2O Aqueous mounting medium METHOD 1. 5. and two baths of xylene (5 min each).13. Cover-slip the slides with permount. Dehydrate the slides by transferring through three baths (2. then into the H2O bath until ready for cover-slipping. Serially transfer 20 µl from each tube to the next tube in the row. then quickly rinse the excess stain from the slides by dipping in water. 50% isopropanol/50% xylene METHOD 1.1.3 Toluidine blue staining (ISH counter-stain) Materials 0. developed ISH slides into the 60% ethanol bath for ≥3 min 2. 4. Transfer the slides through two changes of isopropanol (2. 0. for use in the L929 cell cytotoxicity assay). and add 199 µl of standard dilution medium (DMEM-0% FCS/ActD) to the first and 180µl to the others. Tris-buffered saline (TBS).. 60% ethanol.98 3.5 min each) of isopropanol. one change of 1:1 isopropanol:xylene (2. you will need to perform serial dilutions to generate standard curves. To the first tube. Label a series of eppendorf tubes (in this case. one bath (2. Place the water-washed. 3. 40.g. Transfer the slides quickly through 10% methanol (three quick dips to get rid of excess stain).1. 2. 20 µl of the '40' tube will now contain 40 U TNF).5 min) isopropanol/xylene [1:1]. cytokines) For a number of different assays. appear sufficiently stained. Transfer the IHC-stained slides from H2O into the Gill's stain for 45 sec 2. such that 20 µl of the resulting solution will contain the amount of cytokine needed for your highest standard concentration (here. 5. 4. 1 µl of stock TNFα = 400 U). if not continue staining until the desired intensity of stain is achieved. isopropanol.14 Standard curves (e.2% toluidine blue in 60% ethanol xylene. Briefly rinse the slides in tap water. Depicted is a typical set of dilutions (this one for TNFα. & 0.4.1. 5.
3. You don't need to change pipette tips between tubes.B.004 final. TNF U/well: Vol TNF/eppendorf: Vol. Wash the slides in a 2% solution of organosilane in high-grade acetone for ≈1 min with gentle agitation. 1 µl TNF stock 20µl 40 20µl 4 20µl . even greater adhesiveness can be achieved by treating the dried slides after step 3 with a 10% formaldehyde solution for ≈60 min. If section or cell loss from the slides is still a problem. followed by air drying) . 5.99 performing serial 10-fold dilutions. DMEM/ActD: 40 1 µl 199 µl 4. and sequentially move up the concentration gradient. 1. Since the glass racks are of cast glass. it is one that also is easily overcome by pretreating the slides with TESPA (aminopropyltriethoxysilane or organosilane) Load slides into the glass racks and bake for 3 h at 300˙C. Rinse the slides in high-grade acetone for 1 min and air-dry. To add the standards to the plate.0 20 180µl 3.0 20µl 180µl 0.15 TESPA-treatment of glass slides for in situ hybridization or histology Loss of tissue sections from glass slides during ISH or routine histology can be a real problem. Store the slides at room temperature indefinitely.that means put them in a cold oven and then turn on the heat and also allow them to cool completely before removing them from the oven. Fortunately. so warm and cool them gradually . unless you contaminate a tip in the process. they break very easily with rapid heating and cooling.4 20µl . This way you won't have to change pipette tips for each standard in the series. 2.4 20µl 180µl 0. (N. 4. Each time you add the 20 µl to the next tube.1.04 . start adding the most dilute one to the replicate wells.04 20µl 180µl 0. make sure that you thoroughly mix the tubes before withdrawing the 20 µl for the next step.
Store at room temperature in an amber or foil-wrapped bottle. (the solubility of ammonium sulfate is 76 g/100 ml at 100˙C) Borate-buffered saline For dialysis with purified IgM antibodies. add: 20.5 with 1 M NaOH Add H2O to 1000 ml & filter sterilize ELISPOT & ELISA Carbonate Coating buffer Recipe for 1000 ml To 900 ml of H2O. add: 76 g of ammonium sulfate bring to a near boil allow to cool & then sit overnight at room temperature.56 g NH4Cl pH to 7.2 APPENDIX B -.42 g Tris 7. Recipe for 1 litre: To 900 ml of H2O. add: 2. Autoclave to sterilize.6 g Na2CO3 (>15 mM) 2. add: 1. To prepare acidified isopropanol.9 g NaHCO3 (>35 mM) 0. Actinomycin D is a potent transcription inhibitor that is used routinely in the TNF assay to increase the sensitivity of the L929 cells to the cytotoxic effects of TNF.REAGENTS & SOLUTIONS CELLULAR IMMUNOLOGY REAGENTS Acidified isopropanol. It should be agitated by vigorous pipetting before aliquots are removed for use.76 g sodium chloride pH to 8.5 with NaOH. Alsevers solution Recipe for 1 liter To 900 ml of H2O. add: 5.2 g NaN3 (>3.5 g dextrose (>114 mM) 7.9 g sodium citrate-2H2O (> 27 mM) dissolve the reagents & adjust pH to 6. .100 5. We prepare it as a 5 mg/ml solution (or perhaps more accurately suspension) in 95% ethanol. add 376 µl of concentrated HCl to 100 ml of isopropanol. for use in solubilizing the water-insoluble formazan precipitate formed within the mitochondria of cells cultured in the presence of MTT. Recipe for 1 liter To 900 ml of H2O.1 with 1M citric acid Add H2O to 1000 ml & filter sterilize Ammonium chloride For lysis of red blood cells.72 g sodium borate 8.1 mM) dissolve the reagents & adjust the pH to 9.2 with HCl Add H2O to 1000 ml & filter sterilize Ammonium sulfate (saturated solutions) Recipe for 100 ml: To 100 ml of H2O.
add: 6. PAGE running buffer (5x. > 8.1 mM) 9.15 g Na2HPO4 (anhydrous.0 g NaCl (>154 mM) dissolve the reagents & adjust the pH to 7.23 g NaH2PO4 (anhydrous. > 1. aliquot and store at -70C PAGE gel fix buffer 25% (vol/vol) isopropanol 10% (vol/vol) acetic acid (can be stored indefinitely at room temperature) Phosphate-buffered saline (PBS) Recipe for 1000 ml To 900 ml of H2O.2-7. Tris-glycine) Recipe for 1 litre To 800 ml of H2O . Autoclave to sterilize.0 ml of 0. add: 2.25 ml of PAGE 4x stacking gel buffer 5 ml glycerol 1 g SDS 0. add: .0 g SDS dissolve contents and add H2O to 1 liter (do not adjust pH) store at 4˙C and dilute 5x with H2O before use PAGE 2x sample prep buffer (denaturing.25 mg bromophenol blue add H2O to 25 ml final volume and mix.5 ml of Giemsa stock solution 3.0 ml of 0. Giemsa Stain (prepare fresh each time) Recipe for ≈125 ml To 100 ml of distilled water. It may be convenient for you to make this up as a 10x PBS solution. that can simply be diluted 10-fold and used as is.101 Isotonic Percoll Density Gradient Medium To prepare any amount of isotonic Percoll. but not reducing) Recipe for 25 ml To 10 ml of H2O .4 with 1 M NaOH or 1 M HCl. add: 15.2 M disodium phosphate buffer Briefly stir to mix ingredients before use.1 g Tris base 72 g glycine 5.0 ml of methanol 11. add: 0. Giemsa Stock Solution (for Giemsa stain) Recipe for ≈100 ml To 25 ml of glycerol.1 M citric acid 6.9 mM) 1. mix together: 9 volumes of stock Percoll (purchased from Pharmacia as a 100% solution) 1 volume of 10x HBSS (Ca++ and Mg++-free) This will make the Percoll isotonic with the cells to be fractionated on the gradients.
After dissolution of the EDTA. 2-mercaptoethanol can be substituted.5 M EDTA.61 g of EDTA.. (Tris containing solutions cannot be treated with DEPC. guanidine thiocyanate 0.98 g.0) Add H2O to 100 ml.4% Trypan Blue (in PBS) is a vital dye (i. sarcosine add DEPC-H2O to 98. sodium citrate 0. DEPC-treat and autoclave DEPC-treated water (& other solutions) All water and solutions for use with RNA must be treated with diethylpyrocarbamate (DEPC) before coming into contact with the RNA. Cesium Chloride (for isolation of total cellular RNA) Recipe for 100 ml of CsCl: 95.0 (by adding 10M NaOH). pH 8.0. The dead ones can usually be distinguished morphologically from the live ones.. add ≈90 ml of H2O. to 18.6 ml & adjust the pH to 7.5 g.e.1% final concentration with a pipette and shake the solution vigorously to disperse the DEPC (allow any built up pressure to escape from the vessel periodically.5M). re-adjust pH to 8. is used with live cells) that is not taken up by viable cells.0 To prepare 100 ml of 0.e. To dissolve the EDTA. dilute the cell samples 1:1 with 0. For the washing steps of the ISH protocols.102 3. The nuclei of these cells stain an intense blue colour. Meth Enzymol 154:3-29) . which reacts rapidly with the amines in the Tris) Dithiothreitol (DTT.97 g CsCl 0. Guanidinium Isothiocyanate (GSCN).83 ml 3M sodium acetate (pH 6.4% trypan blue.. pH the solution to>pH 8. just add DEPC directly to the solution to 0. & allow to cool completely before removing the well combs. Kawaichi et al. every minute).39 ml 2-mercaptoethanol and filter sterilize (ref: Okayama.0 add 1. EDTA (0. 1 M) is used in many solutions to prevent non-specific interactions between sulfhydryl groups.5 M GSCN: 64. then allow the treated solution to sit ≈12 h at room temperature or at 37˙C. but is taken up by effete cells. while fully dead cells or live ones take up no dye. It is heavily used for the in situ hybridization protocols employing 35S-UTP probes. For use in hemocytometer counting of cells. To treat a solution.8 g Giemsa powder Heat the glycerol to 60˙C for ≈2 h. 5.64 g agarose dissolve the agarose in the water in the microwave or a boiling water bath & allow the solution to cool to 56˙C.735 g. then autoclave to hydrolyze the residual DEPC. i.5 M Recipe for 100 ml of 5. Store at room temperature indefinitely 0. MOLECULAR BIOLOGY REAGENTS Agarose/formaldehyde/MOPS gel (for electrophoresis of RNA) Recipe for 220 ml gel (scale down as necessary): 137 ml of DEPC-treated H2O 2. then add: 39 ml of formaldehyde 44 ml of 5x MOPS buffer pour the 56˙C solution into the gel casting apparatus. and work the stain into the glycerol (either with a mortar and pestle or stirring) Add 75 ml methanol and continue stirring overnight.
103 ISH 10x salts (store @ -20˚ or -70˙C) To prepare 10 ml. sequentially add to a 15 ml polypropylene centrifuge tube .
although it seems to be better to process tissues to paraffin blocks (by routine methods) than to hold them for prolonged periods in 70% ethanol. Northern blotting pre-hyb/hybridization solution To prepare 30 ml. sequentially add to a 15 ml polypropylene centrifuge tube: 1200 µl of deionized formamide 480 µl of 50% dextran sulfate (to decrease viscosity. add together: 85 ml of 100% ethanol (RNAse-free) 10 ml of 40% stock formaldehyde (use a high grade of chemical) 5 ml of glacial acetic acid briefly mix. and then continue adding the rest of the ingredients 3 ml of 1 M HEPES 1. & use a 5 ml pipette) (vortex vigorously at this point to thoroughly disperse the dextran sulfate) 240 µl of 10X salts (vortex before pipetting) 24 µl 1M DTT 24 µl yeast tRNA (10 mg/ml) 480 µl DEPC-H20 2448 µl (≈ total volume) vortex vigorously again to completely mix the reagents & check/adjust the pH (with paper) to 5.5 .H2O to 1000 ml (200 ml 1M) (16. Harvard Medical School) 100g phenol (heat in a 56˙C water bath to melt the phenol 45.104 ISH hybridization buffer (store @ -20˚ or -70˙C) To prepare ≈2. It stability largely arises from the extra anti-oxidants added relative to many other recipes (recipe.6.02 ml DEPC-treated H2O 11.5 M) Phenol (salt-saturated) this recipe produces a very stable phenol solution. pH 7) (10 ml 0.2 M sodium acetate 50 mM EDTA (pH 8. Dan Tenen.5M EDTA (pH 8.0) 3 ml 50X Denhardt's 3 ml of 10% SDS 1.28 g MOPS powder add DEPC-H2O to 1000 ml.32 ml of DEPC-H2O MOPS (1 M) 231.5 59.5 ml of 10% Na pyrophosphate 2. then store on ice until ready for use.5 ml. Fix 3-6 mm blocks of tissue for 3 h on ice (or cell suspensions for ≈30 min).5 ml of 10 M NaCl 300 µl of 0.6 ml 3M.45 µm filter) 5X MOPS Buffer MOPS 0.0) 5 mM Add DEPC.4 ml 2 M Tris pH 7. then transfer the tissues/cells to 70% ethanol and store indefinitely at -20˚C -.0 (use about 10-12 µl of 1M HCl) ISH fixative (prepare fresh each time) To prepare 100 ml. DNA.s. mix together: 15 ml of deionized formamide 375 µl of salmon sperm DNA (s. and filter sterilize (0.35 ml m-cresol . boiled) (add the hot DNA to the formamide and vortex vigorously to completely disperse it. pre-warm in microwave oven.
8 g NaI 1. add together: 90. To prepare 100 ml.8g sodium acetate . Aliquot and store at -20C. (boil again before use) Sodium acetate (3M) 40. wet with a little EtOH. tie off the ends & drop it into NaI solution (to keep it sodium sulfate-saturated.0) (final .0.5M EDTA (pH 8.4). Salmon sperm DNA weigh out DNA.5 M RNA sample prep buffer (Northern analysis) 5X MOPS 200 µl formamide 484 µl formaldehyde 61 µl H 2O 186 µl denature RNA in sample prep solution for 15' @ 65C RNA sample dye/loading buffer (for visualization of progress during Northerns) glycerol (final . Shear the DNA by forceful passage through a18 ga needle & denature it by boiling for 10 min.4%) 40 mg xylene cyanol (final .5 g Na2SO3 into a piece of dialysis tubing.0.5) General purpose restriction endonuclease buffers (no. low. then add sufficient H2O to bring the DNA to a final concentration of 10 mg/ml.105 454 µl 2-mercaptoethanol 227 mg hydroxyquinolone Reagents for purifying DNA from agarose gels (store in the dark at 4˙C) NaI solution to dissolve agarose gel.5 g Na2SO3 add DEPC-H2O to 100 ml & mix the solution filter through Whatman #1 filter paper and then place 0.1 mM) 20µl bromophenol blue (final .50%) 5 ml 0. 10 mg/ml) Prepare 10 mg/ml RNAse solution in 10 mM Tris (pH 7.0 M .4%) 40 mg DEPC-H2O 5 ml add ≈2 µl of loading solution to each sample RNAse A (heat-inactivated to remove DNAse activity.1 M NaCl 10 mM Tris (pH 7. Ethanol wash solution (store at -20C) 50% EtOH 0. Heat-inactive the DNAse by incubating for 15 min at 70˙C & then aliquot and store @ -20˙C. medium & high salt) reagent Tris/HCl (pH 7.5 M medium 100 mM 100 mM 10 mM 1 1. high 100 mM 100 mM 10 mM 1 1.5) MgCl2 dithiothreitol BSA (mg/ml) NaCl none 100 mM 100 mM 10 mM 1 0M salt level low 100 mM 100 mM 10 mM 1 0. 15 mM NaCl.
1% DEPC. and autoclave .106 H2O. pH > 6.0 treat with 0. qs to 100 ml.
pH to 7.2 g Na citrate 352.5 ml of 2M) (200µl of 0.5M) (1 ml of 10M) qs to 100 ml .9 g add H2O to 3 liters.0. and then H2O qs to 4 liters. STE buffer Tris EDTA NaCl H 2O 10 mM 1 mM 0.1 M (0.107 20X SSC (4 liters) NaCl 701.
To 900 ml of RPMI-0% FCS. For DMEM-5% FCS. we have ours made by GMP. 100 µg/ml streptomycin sulfate. 2 mM L-glutamine. from GIBCO. We generally have ours made up from powder by the Glassware & Media Preparation (GMP) laboratory in the department. This is an alternate general purpose medium. Use the stock GMP RPMI: to 980 ml of RPMI. reduce the amounts of FCS proportionately. However. with bicarbonate buffer and L-glutamine. with bicarbonate buffer and L-glutamine. reduce the amounts of FCS proportionately. from GIBCO. from GIBCO. 1% antibiotic/antimycotic solution (Gibco. RPMI-10% FCS. etc. As with our DMEM. . we have ours made by GMP. To 900 ml of DMEM-0% FCS. and 0.25 µg/ml amphotericin B). stock: 100 U/ml penicillin G. and 10% Con A-stimulated mouse spleen cell-conditioned media) DMEM (Dulbecco's modified eagles medium. A recipe for this reagent can be found in Current Protocols in Immunology. AKA Dulbecco's minimal essential medium). you need to supplement the DMEM stocks with L-glutamine. 10 ml of 5x10-3M 2-mercaptoethanol (and. RPMI 1640 (Roswell Park Memorial Institute medium #1640). As with our DMEM. and increase the amounts of RPMI0% FCS. 5x10-5 M 2mercaptoethanol. 10% FCS. add 10 ml of 100x antibiotic/antimycotic solution from GIBCO (stock: 100 U/ml penicillin G. For RPMI5% FCS. this medium is the same as indicated for DMEM-10% FCS. DMEM-0% FCS.25 µg/ml amphotericin B). which can be purchased either pre-made or as a powder. if the medium is old. 10 ml of 5x10-3M 2-mercaptoethanol (and. 10 ml of 100x L-glutamine). DMEM-10% normal horse serum. add 100 ml of heat-inactivated fetal calf serum. 100 µg/ml streptomycin sulfate.25 µg/ml amphotericin B). In general..3 APPENDIX C -. This is a general purpose medium. RPMI-0% FCS. DMEM-10% FCS. etc. 100 µg/ml streptomycin sulfate. if the medium is old. It is very convenient to purchase this as a 10x concentrated solution. DMEM-10% normal horse serum is used for the L929 cells in the TNF assay.TISSUE CULTURE MEDIA Click's medium. Heat-inactivate by incubating the pre-warmed serum in a 56˙C waterbath for 30-45 min. then after a few weeks of storage. they prepare it with a bicarbonate buffer and Lglutamine. so that it is ready to be used as is. add 100 ml of heat-inactivated fetal calf serum. and increase the amounts of DMEM-0% FCS. which can be purchased either pre-made or as a powder. which can be purchased either pre-made or as a powder. normal horse vs fetal calf). 10 ml of 100x L-glutamine).108 5. and 0. For use in situations wherein you don't want any serum in your medium. Heatinactivate by incubating the pre-warmed serum in a 56˙C waterbath for 30-45 min. for use in hypotonic lysis of red blood cells or in diluting Percoll MEM (minimal essential medium) This is an alternate general purpose medium. HBSS (Ca++ and Mg++-free) This can either be purchased from GIBCO or prepared from scratch. Use the stock GMP DMEM: to 980 ml of DMEM. With the exception that the serum source is different (i. since L-glutamine degrades at 4˙C.e. and 0. For use in situations wherein you don't want any serum in your medium. add 10 ml of 100x antibiotic/antimycotic solution from GIBCO (stock: 100 U/ml penicillin G.
MC/C57. In our hands.MAINTENANCE OF CELL LINES 7TD1 cells (for assay of IL-6). equine. these cells are responsive to bovine. ConA or PHA) in order to respond to IL-1.G4.e.1 cells (C57 mast cells). Pu5 cells always look horribly unhealthy in culture. and are passaged with versene and trypsin. Pu5 cells were one of the lines originally used to clone murine TNFα. Recent molecular phenotyping evidence indicates that these cells arose instead from BALB/c mice. 7TD1 cells are an IL-6 dependent murine hybridoma cell line that can be purchased from the American Type Culture Collection (ATCC #CRL ). L-glutamine. and were selected in part because. LM-1 cells comprise a sub-clone of the IL-1-responsive ATCC cell line D10.MC/C57. and are a very potent source of many cytokines. Cl. 10% normal horse serum.109 5. Pu5-1. .8 cells (ATCC #) in DMEM-10% FCS.8 cells (macrophage cell line) subconfluent monolayer cultures of Pu5-1. TNF will kill these cells with the kinetics observed with natural cytotoxic cells (i. followed quickly thereafter by washing with regular growth medium to prevent over-digestion of the cells. The cells are necessarily maintained at sub-confluent levels (over-growth dramatically diminishes their sensitivity to TNF). The line of L-929 cells that we are using are exquisitely sensitive to the effects of TNFα (ATCC # ).. supplemented with 10% Con A-stimulated mouse spleen cell-conditioned medium. primarily because of the fact that they produce enormous amounts of the cytokine relative to most macrophages or macrophage cell lines. streptomycin. that is best split by minimal trypsin digestion in medium lacking FCS.. fungisone (PSF).e. murine and human IL-6. ≈18 h). They are maintained in Clicks-10% FCS. they do not require sub-mitogenic doses of mitogens (e. transformed murine mast cell line. or they will lose their IL-6 responsiveness. These were the cells originally used to examine the production of many cytokines in mast cells. 7TD1 cells are normally adherent.g. Unlike more cell lines.1. as opposed to natural killer cells (which require much less time to kill their targets). They comprise an adherent cell line. They were sub-cloned by the immunologists at VIDO (U of S). antibiotics. They must be maintained in a subconfluent state (i. L-glutamine.4 APPENDIX D -. They are maintained in RPMI 1640. 10% FCS. L929 cells are maintained in DMEM. They are maintained in DMEM-10% FCS. We maintain our cells in recombinant human IL-6. LM-1 cells (for assay of IL-1).. L-929 cells (for TNF bioassay). 5x10-5M 2-Me and require a source of IL-6. Cl.1 cells are an FcεRI-positive. penicillin. unlike thymocytes. that was ostensibly originally derived from the liver of a C57/Bl6 mouse. ≤1x105 cells/ml). and 5x10-5 M 2-mercaptoethanol.
Binding affinities of Immunoglobulin G binding matrices IgG Binding Matrix SPECIES AVID AL TM HUMAN RABBIT MOUSE GUINEA PIG BOVINE PIG RAT GOAT HORSE SHEEP CHICKEN ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Protein A ++ ++ ++ ++ ++ ++ Protein G ++ ++ ++ ++ ++ ++ + ++ ++ ++ - .5 APPENDIX E -.110 5.
111 5. IL-3. and identification of eotaxin sequence variants. -8. -7. MIP1α. -4. Cytokine mRNA expression patterns in human esophageal cancer cell lines.HUMAN CYTOKINE RT-PCR PRIMERS PCR PRODUCT (BP) 868 533 420 496 493 365 685 462 389 625 606 471 578 207 573 456 340 379 417 260 822 598 CYTOKINE 5' PRIMERS 3' PRIMERS 5'-GGTTTTCCAGTATCTGAAAGTTCAGT 5'-GATCTACACTCTCCAGCTGTAGA 5'-GTTAGTGTTGAGATGATGCTTTGAC 5'-GGCCCAGAGAGAGAGCTGGAG 5'-TCAGCTCGAACACTTTGAATATTTCT 5'-GGTTTACTCTCCGTCTTTCTTCT 5'-TCTGAGGAGAGCGCGGTCGT 5'-TTTTCAGTGTTCTTTAGTGCCCATC IL-1α22 5'-ATGGCCAAAGTTCCAGACATGTTTC IL-1β IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-10 IL-11 IL-13 IRAP 5'-AGCTGATGGCCCTAAACAGATGA 5'-ATGTACAGGATGCAACTCCTGTCTT 5'-AATCCAAACATGAGCTGCCTGCC 5'-TCACATTGTCACTGCAAATCGACA 5'-ATGCACTTTCTTTGCCAAAGGCAA 5'-TATCTCCCCTCCAGAAGCCCAG 5'-TCTTTTAGGTATATCTTTGGACTTC 5'-GCTTCTAGGACAAGAGCCAGGAAG 5'-TCCTAGAGTCTATAGAGTCGCC 5'-ACATGGCTGTGTTTGCCGCCT 5'-AAGCCACCCAGCCTATGCATCCG 5'-GAATTCCGGGCTGCAGTCAC 5'-CTTGGATACCACAGAGAATGAATTTTT 5'-AAGGCATGCACAGCTCAGCACT 5'-TAAGATCTGGCTTTGGAAGGA 5'-GATGCTTTCGAAGTTTCAGTTGAACC 5'-AACAGGCAGGCCTGGGCAGTA 5'TGGCTTTGGAGTTGGAGATTTTTGG 5'-GGAGGGCTTGGCTCAGGGCT 5'-TCCAGCCTCATCGGCCGGT 5'-GGCCACACACTCACCAGCCG 5'-CCCTCAGGCACTCAGCTCTA 5'-GTAAGGGCAGGGACCACCCTG 5'-CTCTACTCGATCCTACCTCT 5'-TTACACTTCAAACTCTCTCTCTTT 5'-TCCCAAAGTAGACCTGCCCAGA eotaxin235'CCCAACCACCTGCTGCTTTAACCTG G-CSF 5'-CTGTGGCACAGTGCACTCTGG GM-CSF 5'-ATGTGGCTGCAGAGCCTGCTGC LIFa 5'-TCTGAAGTGCAGCCCATAATGAAGGT M-CSF 5'-AAAGTGAAAGTTTGCCTGGGTCCT MIP-1α 5'-CAGACAGTGGTCAGTCCTTTC MIP-2β 5'-GCTTCCCGACGCGTCTGCTGA RANTES 5'-GCTGTCATCCTCATTGCTAC SCF TNFα 5'-CTGCTCCTATTTATTCCTCTCGTC 5'-CTTCTGCCTGCTGCACTTTGGA 5'-CCTCGGTTCACAGCACACACTTCAAGA 659 MCP-3 5'-ACCTGCAGATTTATCAATAAGAAAATCCC 5'-CCCGGTCCTGAAATACTTCGTGGACCAGTGGTT 178 22 Τηε προτοχολ φορ ΡΤ−ΠΧΡ αμπλιφιχατιον οφ ΙΛ−1α. and IRAP. & Cytokine Res 15: 1005-09. (1995). SCF & TNFα can be found in Oka et al. mRNA expression. G-CSF. -10. (1996). J Interf. MIP2β. M-CSF. GM-CSF. -5. & -13. IL-1β .6 APPENDIX F -. -6. -11. 23 The RT-PCR protocol for amplifying eotaxin mRNA is available in Bartels et al. LIF. Human dermal fibroblasts express eotaxin: molecular cloning. Biochem Biophys Res Commun 25: 1045-51 .
....au/fimsa.................http://immuno.compuserve.............ac......nih..........htmi Immunoglobulin structure and sequences........gov National Institutes of Health (NIH)...........unige..mrc-cpe...ncbi.........................htmi .......http://www........http://www.112 5..it/cldb/descart5...........umds.......................path..blackci..nim.....................gov National Library of Medicine (USNLM)..gc...........http://www..nsf Federation of Immunological Societies of Asia-Oceania.http://www.....................nih........Imb........http://golgi... .................http://www...............mrc-cpe....................htmi Medical Research Council of Canada ..http://www...................http://www. Asthma and Immunology...........htmi Services provided by individuals’ homepages Cytokines and receptors.....ist..ncbi..... Immunology (medical Specialties)..........edu/others/aal/ Australasian Society for Immunology.........gov/ NCBI WWW Enerez Browser....ac.................htmi American Association of Immunologists..cam.harvard..........ac....uk/ European Collection of Animal Cell Culture (ECACC).......................................nwu.............................................nim....................nim..ucl................uk/society/bsi/ CanadianSociety for Immunology...com/JI Kabat Database of Sequences of Immunological Interest..uk/ Tissue typing.............htmi Cytokines and receptors.............com/hema/ National Academy of Science (US)..http://www....bbk..............ac.......edu....http://www...einet.......http://www.....................kcj.......uk/elsewhere/tissue/whatl..wehi...... http://www....7 APPENDIX G -.cam........................ http://www....................................ncbi...........htmi Jackson Laboratories (source of mice)…………………………………..........nlm..... ...............................ucalgary...jax......html Immunogenetics database (IMGT Database)..de/groups/ibelgaufts/cytokines.ox.......... .........atcc........http://www.....htmi MHC structure and fanction.........htmi The World Wide Web Virtual Library: Immunology (Biosciences).......http://www.......au/societies/ASI/ASI....................nas......gov/Entrez/index.........................ac.http://www.......edu.................http://ourworld............uk/contrib/imgt/top-imgt.................html Society homepages American Association of Allergy...............co.......ac..http://execpc.http://www3...at-home...................net Centre for Protein Engineering: Home Page (MRC-CPE)....http://www.http://www... http://www....htm International Society of Experimental Hematology..http://ji....edu/biopages/immuno.bme..ca/csiweb.......csi....ocms...biochem...uk/~mrc7/mikeimages............nih... http:/histo...edu Linscott's Directory of Immunological and Biological Reagents.............htmi Vbase: a Directory of Human Immunoglobulin V Genes.....http://glamdring.http://www................ca/title...adelaide.............................gov/Web/Search/index......org/ Bionet Electronic Newsgroup Network for Biology (BIOSCI)...............ucsd........ebi........cryst......microbiology................ac............ http://galaxy........................... http://www...com/homepages/LINSCOTTSDIRECTORY/ National Center for Biotechnology Information (NCBI).nih.......bio..uk/~smb/cyt_web/ Immunoglobulin structure and function....edu Online services and databases American Type Culture Collection (ATCC)...uk/~martin/antibodies....uni-muenchen.......cam...........net/galaxy/Medicine/MedicicalSpecialties/Immunology....................com/~edi/aaaai........ac..journals.........http://www..................org/jaxmice Journal of Immunology.....http://www..htmi GeneBank (Searches address)...http://www.htmi British Society for Immunology.....nih.....mrc........uk/imt-doc/vbase-home-page...... CD antigens...............................WORLD WIDE WEB Immunology sites of interest The following comprises a list of potentially useful web sites for immunologists............
and Haemophilus somnus IgBPs FD Bastida-Corcuera. LB Corbeil pp 143-149 Binding of bovine IgG2a and IgG2b allotypes to protein A. LB Corbeil . protein G.113 Veterinary Immunology and Immunopathology 71: (1999) pp 115-123 Differential complement activation by bovine IgG2 allotypes FD Bastida-Corcuera.
114 5.SELECTED TEMPLATES FOR 96--WELL PLATES 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 1 A B C D E F G H 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12 .7 APPENDIX G -.
115 1 A B C D E F G H 1 A B C D E F G H 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12 .
116 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12 .
50 .1 INDEX L-929 cells 23 lauryl sarcosine 60 10xSSC 63 32P-labelled cDNA probe synthesis 66 5X MOPS 63 7TD1 cells 21 actinomycin-D 23 activation of macrophages 16 ammonium persulfate 38 anaesthetic 8.5 (anti-CD4) 31 GSCN lysis solution 60 H2O-saturated isobutyl alcohol. 100 antibody-dependent phagocytosis 14 antibody-coated cells 14 ADCC 14 anticoagulant 8 AVID-AL IgG affinity columns 32 blast assay 46 bromophenol blue dye 39 C'-dependent cytotoxicity 42 capture antibodies 50 cell counting 96 cellular RNA 60 cellular viability 97 Concanavalin A 46 CsCl/sodium acetate 60 cytocentrifuge preparations 97 cytotoxicity assay for TNFα 23 density gradient systems 8 detection antibodies 50 detection of mRNA bands 70 ELISPOT 50 ELISPOT plates 50 fluorescein-labelled anti-CD4 42 Giemsa stain 57 Gill's hematoxylin 102 GK1. 12. 38 hemocytometer 96 heparin 8 IgE anti-DNP antibody 29 IL-1 activity 18 IL-6 activity 21 immunohistochemistry 58 in situ hybridization 72 intradermal skin test 56 isopropanol fix 39 isotonic Percoll 9 leukocyte/platelet-rich plasma 9 lipopolysaccharide 16 LM-1 cells 18 low-tox rabbit C' 42 LPS 16 Lymphocyte separation medium 50 mast cells 29 monoclonal antibodies 31 monocyte/macrophage monolayers 14 monokine bioassays 18 morphologic identification of PBL 101 MTT 18 neutrophil chemotaxis assay 25 neutrophils 8 nitrocellulose 40 Northern blot blocking solution 68 Northern blotting 60 Northern blotting apparatus 63 PAGE 2x sample prep buffer 91 PAGE running buffer 91 paraffin section 57 paraformaldehyde 43 PBST 50 percoll 9 peripheral blood 8 phycoerythrin-labelled anti-CD8 antibodies 42 platelet 9 PMN 8 polyacrylamide gel electrophoresis 38 polymorphonuclear cells 8 pre-hybridization (Northerns) 68 radioactive hybridization solution 68 rapid Coomassie blue stain 38 rapid Coomassie brilliant blue 39 RBC sedimentation buffer 9 recombinant human IL-6 21 RNA sample dye/loading solution 63 RNA sample prep solution 63 rouleaux 9 separating gel (PAGE) 38 stacking gel (PAGE) 39 streptavidin-AP conjugate 40.
2 Synthesize the cRNA probes 72 T cell responses 56 Th1 versus Th2 responses 56 TIB 211 (anti-CD8) 31 tissue processor 57 TNF activity 24 TNFα standards 23 two-colour FACS 42 unit of TNF activity 24 web sites for immunologists 105 Wrights-Giemsa staining 101 x-ray film 70 Zeta-bind transfer membrane 63 .
The blot was probed with a 32P-TNFα cDNA and washed at high stringency (Gordon & Galli. bottom left .Antigen-driven IFNγ and IL-4 production in splenocyte cultures from BALB/c mice vaccinated (dy 0) and boosted (dy 14) with a range of doses of ovalbumin (0.10 µg) in the context of alum (taken from Schneider & Gordon. Ph.1 A PRACTICAL GUIDE TO CELLULAR AND MOLECULAR RESEARCH METHODS IN IMMUNOLOGY John R. John R. top right .ca --------------------------------------------------------------------------Front cover: top left . 1994). The tissue was harvested at 8 h post-challenge and was probed with a 35S-α1 (I) collagen cRNA probe. J Exp Med 180: 2027.Northern blot autoradiograph of mRNA from Cl.usask.In situ hybridization autoradiography of mouse skin tissues undergoing a passive cutaneous anaphylaxis response following intravenous allergen challenge. University of Saskatchewan.D. which hybridizes specifically with fibroblasts activated by mast cell TNFα and TGFβ (Gordon & Galli. manuscript in preparation) . FAX: 306-966-7244 email: gordon@sask. Saskatoon. CANADA SECOND EDITION © 1998. Gordon tel: 306-966-7214. J Exp Med 174: 103.05 . Gordon.MC/C57. Department of Veterinary Microbiology.1 mast cells challenged via the FcεRI for varying periods of time. 1991).
and moderate levels of RANTES & GM-CSF (Hull. manuscript in preparation) . The tissues contained high levels of MCP3.2 bottom right . Saxena & Gordon.Chemotaxis assay of the eosinophil chemotactic activities of extracts from the skin of an eosinophilic epitheliotropic T cell lymphoma patient.
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