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EXERCISES IN CELL BIOLOGY FOR

THE UNDERGRADUATE
LABORATORY
A PROJECT OF THE AMERICAN SOCIETY FOR
CELL BIOLOGY EDUCATION COMMITTEE, 1992
Compiler: J udith Snyder
Editor: Mary Lee S. Ledbetter
EDITOR'S INTRODUCTION
This collection of laboratory exercises represents the efforts of a number of members of the
American Society for Cell Biology. It arose from discussions of the current crisis in science
education in general, and in particular the declining interest in and preparation for careers in
scientific research among American undergraduates. Recent studies have pointed to laboratory
experience as central in motivating undergraduate students to continue their scientific education.
Though the most successful experiences are those in which the student actually conducts
independent research under the supervision of a faculty mentor, more formal "investigative"
exercises are also effective in engaging student interest.
Very few resources are available to faculty members attempting to implement such an approach
in their own laboratory teaching, particularly in areas beyond their own research expertise. Most
commercial textbooks designed for undergraduate use in a comprehensive introductory cell
biology course emphasize descriptive approaches and a "cookbook" style, so that the student
may encounter the material rather passively. Particularly in cell biology, an additional difficulty is
the dependence of modern experimental approaches on advanced instrumentation and
expensive reagents, both beyond the means of many undergraduate programs, if they are to be
available to more than a handful of students. Nevertheless, examples can be found of imaginative
exercises designed with the goal of engaging the student in active consideration of fundamentally
important cell biological principles, and capable of being accomplished even under the constraints
of time, money, and faculty expertise that prevail in undergraduate settings.
An informal network over the years has promoted sharing of such ideas among like-minded
faculty members. The Education Committee felt that the enterprise would be served by
attempting to formalize the process and to provide a means of distribution of effective ideas. The
Committee therefore solicited contributions from the membership through an announcement in
the Newsletter and by requests to those known to be on the grapevine. These are among the
early contributions. They have been edited into a common format that includes pedagogical
material (literature references, introductory material, procedures, questions for the students to
consider, suggestions for handling numerical data, ideas for supplementary work) as well as
practical points (time required, lists of needed equipment and supplies, recipes, trouble- shooting,
general level of student background assumed.) Each exercise is the work of the named author.
The text has been examined closely by the editor and others, and changes made with the
authors' approval, but the author is the one with experience using the procedures in a teaching
setting, and inquiries should be directed to him or her.
As a group the exercises are not intended to provide a comprehensive introduction to the field of
cell biology. Indeed the distribution of topics is clearly skewed toward certain areas, as reflects
the interests of those contributing. All do, however, demonstrate significant cell biological
principles, modern quantitative and analytical approaches, or unusual experimental systems.
Frequently effective teaching exercises are adapted from published sources and from the
unpublished work of colleagues. We have made a serious effort to acknowledge such sources,
but inevitably the line between the original and the borrowed may become obscured. Thus any
copyright that pertains to this collection rests with the authors. The collection itself should be
considered more a "circulating library" than a formal publication.
We hope that as time passes, the collection will be expanded and modified to reflect the
experience of users and to include new ideas both of those directly involved in undergraduate
education and of others who may think of ways to demonstrate to undergraduates principles
important in their research. Through such efforts we all will benefit. The standard format for such
submissions is the one used in these exercises. Please send them to me or to the National Office
of the ASCB. To make the collection as accessible as possible, it is therefore being made
available at cost in several formats: photocopy, diskette (either 5.25" or 3.5", formatted for several
common word processors) and E-mail. To obtain a copy, contact the National Office with details
of the format you need.
I am pleased to acknowledge the help of the following reviewers of this collection in its draft
stage: Carole Browne (Wake Forest University), Andrew Dudley (Genentech), Kay Greene (Regis
College), Deborah Kaska (University of California at Santa Barbara), Dawn Larson (University of
Guelph), Tom MacRae (Dalhousie University), George Shiflet (Wofford College), and Ron
Tombes (Clemson University). David Kirk (Washington University) and Roger Sloboda
(Dartmouth College) of the ASCB Education Committee also contributed helpful suggestions.
Mary Lee S. Ledbetter
Worcester, Massachusetts
J une 1992
TABLE OF CONTENTS
I. INTRODUCTORY
A. Microscopy
B. Measurement of cell protein content
C. Cell behavior: mating, phagocytosis and exocytosis in Tetrahymena
D. Cell culture
II. INTERMEDIATE
A. Purification of mitochondria by differential sedimentation and monitoring of cell fractions for
specific activity of succinate dehydrogenase
B. Cell motility: muscle
C. Cell motility: ciliary regeneration
D. Preparation and reactivation of ciliary cytoskeleton
E. Cytoskeletal transformation of sea urchin coelomocytes
F. Cellular immunology: lymphoid organs and the structure and distribution of their cells
G. Cellular immunology: use of surface markers to discriminate T and B lymphocytes
H. Cellular immunology: mitogenic stimulation of lymphocytes
I. Cellular immunology: enzyme-linked immunosorbent assay (ELISA).
INSTRUCTIONS FOR SUBMISSION OF NEW
EXERCISES TO THIS COLLECTION
We welcome additions to this collection of laboratory exercises. Of particular interest are
exercises that require little specialized instrumentation, demonstrate clever techniques and/or
new areas of cell biology, and can be easily accomplished in a typical afternoon laboratory
period. Other sorts of ideas are also appropriate, however, especially if they lend themselves to
undergraduate research projects. If you have exercises that you have found particularly effective,
please submit them to
National Office
American Society for Cell Biology
9650 Rockville Pike
Bethesda, Maryland 20814
If you provide a diskette of a standard word-processing file of the exercise along with your hard
copy, editing will be facilitated. Also please follow the standard format:
TITLE: Brief but descriptive
AUTHOR(s): Names and mailing addresses, so users can contact you with questions
PURPOSE: A brief statement of the principles illustrated in the exercise and/or the techniques
demonstrated.
TIME REQUIRED: as close an estimate as can be made. If this exercise is customarily used in a
teaching lab setting, point out any need to make observations at other times than the normal lab
period.
LEVEL: clearly subjective, but consider whether this would normally be taught to beginning
students (introductory), second- or third-year students (intermediate), or well trained students
(advanced).
REFERENCES: Provide two or three references, preferably to the scientific literature, though
textbooks are satisfactory.
BODY OF THE EXERCISE: This part should be written in a form suitable for use by students with
only minor adaptation.
Introduction: Provide background on the theoretical and conceptual aspects of the exercise and
comment on general principles underlying techniques to be used.
Procedure: This is the step-by-step part students follow to carry out the exercise. It is preferably
not too cookbook, but should invite the students to formulate hypotheses and make decisions and
judgments as they go along. If hazardous materials or techniques are to be used, be sure to
highlight them and draw the students' attention to appropriate precautions.
Data analysis (if appropriate): Suggestions as to how to handle numerical results (graphing,
application of formulae, etc.)
Suggestions for further study: Brief comments as to additional approaches with the same
procedures or alternative procedures to address the same questions are included here. The idea
is that this could be a resource for student projects.
FOR THE INSTRUCTOR: This part is designed to help an instructor unfamiliar with the system to
implement it in his or her laboratory. It should be as detailed as possible.
Equipment: Specify all major facilities and equipment needed and its degree of refinement, e.g.
compound microscope, preferably equipped with phase contrast.
Supplies and sources: Provide ordering information for unusual biochemicals or living materials.
Recipes: Provide instructions for preparing the solutions used in the laboratory. Be careful to note
where the order of mixing is important, where sterility must be by filtration rather than autoclaving,
and how solutions should be stored (frozen, refrigerated, shelf-life, etc.)
Common problems and their likely cause: It is expected that any exercise submitted will work at
least most of the time in your laboratory. Experience usually reveals subtle pitfalls that can be
avoided with knowledge of their existence. This section should include a summary of where those
pitfalls may lie in your experience. If the exercise is unusually free of pitfalls, that can also be
noted.
Microsoft Word
WordPerfect
Title: I. A. Microscopy
Author: Jason Wolfe, Department of Biology, Wesleyan University, Middletown,
Connecticut
Purpose: To introduce students to the light microscope as an observational and analytic
tool in cell biology
Time required: 3 hours Level: Introductory
References: Many standard textbooks of cell biology have excellent introductions to
optics and the principles of light microscopy.
Introduction:
The light microscope is perhaps the single most important instrument used in Cell Biology. It is
used under bright field conditions to study the organization of cells in fixed and stained sections of
tissues. With phase contrast optics it is possible to monitor the movements of living cells and to
observe changes in their subcellular organization. The light microscope may also be used to
monitor certain operations such as cell fractionation and biochemical characterization of cellular
components.
This exercise involves the use of the light microscope under both bright field and phase contrast
conditions. A variety of different cells will be observed. The microscope will also be used to
quantitatively determine the number of cells in a suspension using a special device known as a
hemocytometer, or cell counting chamber.
Procedure:
A. The anatomy of the microscope
The light microscope consists of an optical system and a body. The body is composed of a base
which rests on the desk, a stage which supports the material to be viewed, and a tube through
which the specimen is viewed.
The optical system begins with a light that shines through the condenser lenses located beneath
the stage. The function of the condenser is to focus the light source onto the specimen on the
stage. Light travels through the specimen to the objective. This lens magnifies the image of the
specimen which is carried to the eyes by the oculars or eyepieces.
B. How to focus a microscope
1. With a piece of lens paper, clean the oculars and objectives.
2. Slide the lower power objective into place.
3. Prepare a slide with a drop of liquid containing a specimen, and place a coverslip on
the drop.
4. Transfer the slide to the microscope stage.
5. Adjust the condenser all the way up.
6. Set the condenser on bright field.
7. Watching from the side and being careful not to drive the objective into the coverslip,
lower the objective as far as you can.
8. Viewing through the ocular, focus up (raise the objective) until a semblance of the
specimen is seen.
9. Close the iris diaphragm until it is opened about 1/4 of an inch.
10. Now lower the condenser until the image of the diaphragm is in view. Open the
diaphragm until its image coincides with the boundaries of the field.
11. With a bit of fine focusing you're all set.
C. How to adjust for phase contrast
1. Set the condenser so that its phase ring matches the objective.
2. Remove one ocular and insert the centering telescope.
3. Observe the dark and bright annuli (rings).
4. Center the condenser annulus (bright ring) until it coincides with the objective annulus
(dark ring).
5. Remove the centering telescope and replace the ocular.
D. Using the microscope
One should take great care in handling the microscope. When moving it from one place to
another, it should be gripped tightly with both hands.
The lenses should be repeatedly cleaned with lens paper.
Remember that the image is inverted. When you move the slide to the left, your image will move
to the right.
Take great care in switching from a low objective to a higher objective lest the lens hit the slide.
Always raise the objectives before inserting the hemocytometer. It is much thicker than any
ordinary microscope slide.
Because of the thickness of the hemocytometer, it will probably need a special adjustment of the
condenser position and of the phase rings.
When putting the microscope away, leave the lowest objective in place.
E. Measuring the field size
It is possible to estimate the size of an object by comparing it with the diameter of the field of
view. To do this, it is first necessary to measure the size of the field.
1. With the 10x objective engaged, place a short, transparent ruler over the opening in the center
of the stage so that the lines are visible through the microscope.
2. Move the ruler so that a vertical millimeter mark is just visible at the left edge of the circular
field of view.
3. Count the number of millimeters from the left side to the right. If the right side of the field does
not line up with one of the vertical markings, estimate the fraction of a millimeter. This is the
diameter of the low power field of view. Record your measurement in millimeters (mm) and in
micrometers (um).
4. Carefully move the 40x objective into place. Note that the diameter of the field is less than 1
mm. Rather than measuring the field directly, we can calculate the diameter based on the direct
measurement of the low field diameter and the following equation:
diameter of the high power field

low power magnification
------------------------------------------ = ----------------------------------
diameter of the low power field

high power magnification
What is the diameter of the high power field in millimeters? in micrometers?
F. The hemocytometer
In many cases it is important to know how many cells one has in a suspension. One way to
quantify cells such as bacteria is to serially dilute a liquid culture and spread known amounts of
each dilution on a plate of nutrient agar. Each colony of bacteria may be assumed to have arisen
from a single cell. By counting the colonies and multiplying by the dilution factor, you can
calculate the number of cells in the original culture. A major drawback of this technique is that it is
very time consuming: the plates of bacteria must be incubated overnight before the colonies are
large enough to be seen. An alternative, and one that is applicable to all types of cells, including
cells like erythrocytes which do not divide, is to use a special device called a hemocytometer.
Each hemocytometer has two separate counting areas. Each counting area has 9 large squares
(see Figure I.A.1). In the corners are large squares with 16 small squares (labeled W); on the
sides are large squares with 20 smaller rectangles; the large square in the center has 25 small
squares, each with 16 still smaller squares.
A special coverslip is supported above the grid such that the volume above each large square is
equal to precisely 10
-4
ml (0.1 ul). If 30 cells were located in one large square, then your sample
would contain 30 x 10
4
cells/ml.
The trick in using a hemocytometer is in getting an even distribution of cells over the squares. To
do this, first the hemocytometer with its coverslip in position is placed on the microscope stage. A
drop of evenly suspended cells is then introduced to the edge and fluid is drawn in by capillary
action. If the volume is too great and the fluid flows into the gutters, it cannot be used. If it takes
more than one application to cover the grid, it cannot be used. If the drop is held too long before
applying it to the hemocytometer, it cannot be used.
To check on accuracy, the numbers from several squares are obtained. If they are close, they
can be averaged. If the numbers are far apart, the hemocytometer should be cleaned and
reloaded. A comparison of the numbers from several loadings should be made.
G. Examination of two cell types
Prepare slides of Tetrahymena and E. coli and examine the cells in the microscope, according to
the directions provided.
Tetrahymena is a freshwater ciliate about 50 um long. The surface of each cell is covered with
hundreds of cilia arranged in longitudinal rows which propel the cell by rhythmic beating. Place a
drop of the culture on a clean slide and cover with a coverslip. Observe the cells first under phase
contrast to examine the swimming motion. Touch a piece of filter paper to the end of the coverslip
to remove some water. Can you see the cilia? Can you see the slow pulsation of the contractile
vacuole? The nuclei may be visualized in phase contrast, or by staining the cells in one drop of
acidulated methyl green. How many nuclei can you see in each cell?
E. coli is one of the normal bacteria present in the intestines of warm-blooded animals. E. coli is a
rod-shaped bacterium, or bacillus, that grows as a single cell, but may form chains as cells divide
and fail to separate completely. E. coli, one of the simplest organisms, is one of the most studied
and best understood on a molecular level. Place one drop of the culture on a slide, and observe
under phase contrast optics with the higher power (40x) objective. Can you see any internal
structure in the cells?
H. Determination of cell densities
Your instructor has 3 flasks of E. coli and 3 flasks of Tetrahymena, each with a different cell
density. Select one flask of each cell type and record its identification number. Transfer 1 ml of
each cell type to different test tubes, and fix the Tetrahymena by adding 1 ml of formaldehyde.
(NOTE: Do NOT pipette by mouth.)
First count the Tetrahymena. Position the hemocytometer on the microscope stage and adjust the
objective so you are focused on the grid lines. With a Pasteur pipette and rubber bulb gently
resuspend the cells and transfer a drop to each side of the hemocytometer. Count the number of
cells in the 4 A squares plus the central C square and average them by dividing by 5. How do the
numbers of the two sides compare? If they are far apart, repeat the count after loading the
hemocytometer again. Each value, multiplied by 10
4
gives you the cells per ml. But now you must
multiply by 2 to correct for the two-fold dilution which occurred during fixation. Check with your
instructor to see if your cell density measurements are accurate and calculations correct.
Now count E. coli. Remember, the cells are much smaller and more numerous than
Tetrahymena. There may even be too many to count in a large square, but you can count smaller
squares and then calculate the number that would be in a large square. Or you can dilute your
sample, remembering to correct for the dilution. Check with your instructor to see if your
measurements are accurate and your calculations correct.
J . Examination of other cell types
Three additional cell types are available for detailed observation with your phase contrast
microscope. Look at each and carefully record what you see by sketching - and labeling - in your
notebook. Examine the cells by both direct illumination and phase contrast, low and high power.
There are any number of features you may notice: How big are the cells? Are they flat or do they
have depth? What shape are they? Is there a cell wall? Can you see the nucleus? How big is it
relative to the cell volume? Can you see any organelles in the cell? What do they look like? Is the
cell colorless or colored? If the sample you examine shows more than one cell, are all the cells
the same? How are they different?
1. Squamosal epithelial cells from the cheek
Scrape the inside of the cheek with the flat end of a toothpick. Place on a slide and add a
coverslip. Under low and high power, identify the nucleus, the darkly staining nucleolus,
the nuclear membrane and cell membrane and the cytoplasm.
Look also for bacteria! How many different types do you see? Are any adhering to the
cheek cells?
2. Elodea
Place a young leaf from the tip of the plant in a drop of water on a clean slide. Orient the
leaf so that its upper surface faces you. Cover with a coverslip and examine at low and
high power.
Remember that the cells have depth; try to focus up and down on an individual cell in
order to explore its three-dimensional structure. Note the large central vacuole. The
nucleus is near the cell wall and sometimes difficult to detect. The cytoplasm contains the
chloroplasts which are moved by cyclosis, or cytoplasmic streaming. carefully observe
the pattern of movement of chloroplasts using both bright field and phase contrast optics.
Which cells have more chloroplasts, the upper layer or the lower layer? Can you think of
a plausible explanation?
3. Oscillatoria
Oscillatoria, like E. coli, is a prokaryote. However, it is a blue-green alga (cyanophyte),
capable of photosynthesis, not a bacterium. Under bright field do you see any pigment?
The long unbranched filaments of Oscillatoria are often attached at one end and display
an oscillating motion, from which this form derives its name. Unattached filaments move
by a gliding motion. Carefully observe the motion of the cells in phase contrast.

Suggestions for further study (Editor's addition):
1. Cheek epithelial cells can be stained in various ways before viewing. Methylene blue clearly
reveals the nucleus and (in females) the Barr bodies.
2. Various specimens can be examined in wet mount. Pond water is always lively and pure and
mixed populations of protozoons and algae may be purchased at moderate cost from various
biological supply houses.
3. A standard compound light microscope can be conveniently modified to give dark field viewing
by placing a dime or a quarter (depending on the objective lens) on the condenser lens and
adjusting the condenser position to block the incident light from the objective lens. Polarizing light
microscopy can be approximated using inexpensive polarizing film (from Edmunds Scientific
Supply). One piece placed on the illuminator and a second taped to the ocular lens can
significantly enhance the detail of specimens with orderly structure, such as skeletal muscle or
cilia.
For the instructor:
EQUIPMENT
SUPPLIES AND SOURCES
RECIPES
COMMON PROBLEMS AND THEIR LIKELY CAUSE
Title: I. B. Protein assay and measurement of protein content of cells
Author: Jason Wolfe, Department of Biology, Wesleyan University, Middletown,
Connecticut 06457
Purpose: To demonstrate the principles of colorimetry, serial dilution, biochemical assays,
and cell protein content through the Bradford assay for protein
Time required: 1 or 2 3-hour periods, depending on student preparation
Level: Introductory
References: Bradford (1976) Analytical Biochemistry 72: 248-254
Part A: The protein assay
Introduction:
It is often necessary to determine the concentration of protein in dilute solutions. A sensitive yet
simple colorimetric assay that has gained wide usage was developed by Bradford. The method
involves mixing a dye, called Coomassie Brilliant Blue, directly with a protein solution. The dye
forms a complex with protein. When the dye binds to protein it changes color, which means that it
absorbs light at a different wavelength. The absorption maximum is shifted from 465 to 595 nm.
With a spectrophotometer (see Appendix) it is possible to quantify color by measuring the amount
of light that the solution absorbs. At 595 nm absorption is linear over a specific and narrow range
of 10-100 ug protein.
A standard curve provides a reference for measuring the amount of protein in a solution of
unknown concentration. It is constructed by measuring the absorption of several known
concentrations of protein in the range of 10-100 ug. Then, when a solution of unknown
concentration is measured, its absorbance at 595 nm can be compared to the standard curve.
The trick is that the concentration of the unknown must be in the range of the standard curve, that
is somewhere between 10-100 ug. The dilemma, however, is that the concentration of the
unknown is not known.
A useful strategy for dealing with unknowns is to prepare serial dilutions. Because the linear
portion of the standard curve extends over a full order of magnitude of concentration, a fail-safe
approach for a serial dilution of the unknown is one based on the order-of- magnitude dilutions.
This exercise involves two parts. First, a standard curve will be constructed using a solution of
known concentration of bovine serum albumin (BSA). Then, using the strategy of serial dilution,
the concentration of a mystery solution of BSA (labeled X, Y or Z) will be determined.
Procedure:
1. The standard curve
Obtain a solution of protein whose concentration is known. Bovine serum albumin (BSA) has
been prepared in SDS/EDTA (see solutions below) at a concentration of 1 mg/ml. Dilute 0.5 ml of
BSA with 4.5 ml of 0.01% SDS / 0.1 mM EDTA to yield 5 ml at 100 ug/ml.
Label a set of test tubes (13 x 100 mm) from 1-6 using a black Sharpie marker or pen on tape.
Following the chart below fill the tubes sequentially with BSA, SDS/EDTA and Bradford reagent.
Bradford reagent contains a dye, Coomassie blue, which binds to protein. The dye/protein
complex produces a blue color whose absorbance is directly proportional to the protein
concentration.
NOTE: It is important to mix the tubes rapidly and thoroughly immediately after the dye is added,
one tube at a time. Because the Bradford reagent contains phosphoric acid, avoid contact with
the mouth or skin.
Tube 100 ug BSA SDS/EDTA Brad Reag Prot. A595
---------------- ---------------- ---------------- --------------- ---------------- ---------------

(ml) (ml) (ml) (ug/tube)

1 0 1.0 4

2 0.2 0.8 4

3 0.4 0.6 4

4 0.6 0.4 4

5 0.8 0.4 4

6 1.0 0 4

Enter the amount of protein each tube contains in the column headed "ug/tube."
Full color development should occur in 5 minutes. Assay the tubes with the spectrophotometer
within 1 hour. Use the tube #1, the BLANK, to set the absorbance at 595 nm to zero. Measure the
A595 of each of the other tubes. Write the values in the chart above.
Plot your data on graph paper, ug protein on the X axis and A595 on the Y axis. Show your graph
to the instructor. If a linear graph is not obtained, repeat the assay being more careful with
pipetting.
2. Assay of the unknown
In this portion of the laboratory you will determine the concentration of an unknown solution of
BSA in SDS/EDTA. Dealing with unknowns, determining as much information about them as
possible, is what makes science so much fun. However, the enjoyment factor is nearly canceled
out by the frustration level if the investigation is not carried out in an orderly fashion. For example,
the way to ascertain the concentration of BSA in the unknown is to compare its absorbance to the
standard curve created in the first part of this exercise. You might guess that a five-fold dilution
might do the trick. However, if the A595 does not fit the curve, it will be necessary to dilute the
sample again. If the absorbance reading still does not fit, you would have to try yet another
dilution. This "shot in the dark" technique not only takes a lot of time but uses up quite a bit of
sample and provides plenty of opportunity for error.
The most efficient way to determine the concentration of an unknown is by using the strategy of
the serial dilution. By serially diluting the unknown by an order of magnitude (10-fold) each time,
you will be guaranteed to have one tube with an absorbance value that fits on the standard curve.
Once the concentration of unknown is determined for a particular tube, it is only a matter of a
simple "back calculation" to determine the amount of protein in the original sample.
Label a set of test tubes 1-4 and pipette 0.9 ml of SDS/EDTA into each tube. To the first tube add
0.1 ml of unknown for a total of 1.0 ml. Tube 1 now contains a 10-fold dilution of the unknown or
an order of magnitude less protein than the original tube. Mix the tube gently by pipetting up and
down; try to avoid creating bubbles. This action also rinses the walls of the pipette so that any
solution remaining in the pipette will be of the same protein concentration as the tube. Why is this
important?
Transfer 0.1 ml from tube 1 to tube 2; mix gently. The second tube now contains a 10- fold
dilution of the protein in the first tube, or 2 orders of magnitude (10 x 10) less protein than the
original (Co x 10
-2
). Get the idea?
Now transfer 0.1 ml from tube 2 to tube 3, mix gently. Transfer 0.1 ml from tube 3 to tube 4. How
many orders of magnitude less than the original do you have in this last tube? Express it as a
fraction. Express it also as an exponent. What volume of solution would you have ended up with if
you had made this dilution directly from the original sample? In other words, starting with 0.1 ml of
unknown, how much SDS/EDTA would be necessary to achieve this same degree of dilution
without using the serial dilution technique?
Remove 0.1 ml from tube 4 and discard it (i.e. blow it into the sink). What is the reason for this?
Add 0.1 ml of water and 4 ml of the Bradford Reagent to each tube and measure the A595. Use
the tube with the "best fit" to the standard curve for the back calculation. For example, if the
amount of protein in tube 2 is 35 ug (in a 1 ml volume), then the concentration in the original tube
is
10 x 10 x 35 =3500 ug/ml =3.5 mg/ml
Part B: Use of the protein assay to monitor cell growth
Introduction:
It is frequently necessary to determine how much protein a cell contains in order to learn
something about the characteristics of the cell or to compare one cell type to another. Once that
value is obtained one can use the amount of protein in a cell lysate as a measure of the number
of cells lysed. It is possible to measure the amount of protein per cell using the Bradford
procedure providing you have enough cells to produce more than 10 ug of protein and providing
the number of cells if known. The number of cells in suspension can be collected by
centrifugation, lysed in SDS and assayed for protein colorimetrically.
Procedure:
1. Cell growth.
Tetrahymena cells were grown in culture dishes containing agar with enriched proteose peptone
medium, at room temperature. Cells were washed from the dishes and suspended in 10 mM Tris,
pH 7.4 containing 1 mM CaCl2.
E. coli were grown in nutrient broth at 37
o
C and were suspended in phosphate buffered saline
(PBS)
2. Cell density.
Record the volume of cells of each type obtained. Transfer 0.2 ml of E. coli to dispo tubes, and fix
with 1.8 ml of buffered formaldehyde (10%). Serially dilute (twice) in formaldehyde. Fix
Tetrahymena with an equal volume of formaldehyde.
Using the hemocytometer, determine the density (cells/ml) of each cell type, starting with the
highest concentration of fixed cells. For the Tetrahymena use all nine large squares. For E. coli
use 1 large square (or less). If the cells are too numerous to count, try the next diluted sample.
One calculates the density of cells in the fixed sample by multiplying the average number in one
large square x 10
4
. Then, by correcting for dilution with fixative, one obtains the cell density in the
original sample. Finally, since density is given as cells/ml one can calculate total number of cells
by multiplying the density x the volume of cells.
3. Collecting cells by centrifugation.
Two different refrigerated centrifuges will be used to collect the two different cell types.
Tetrahymena cells will be collected in the low speed IEC at 2,000 rpm for 5 min, using a swing
out rotor. E. coli will be pelleted with the high speed Sorvall centrifuge, operating at 12,000 rpm
for 10 min using a fixed angle rotor. In each case pellet cells from a 10 ml sample will be used.
Note: Whenever the centrifuge is used, the rotor must be balanced with tubes on opposite sides
containing equal volumes. Check that adapters are balanced also. The centrifuge must be
operated with the lid closed tightly. Keep fingers away from spinning rotor.
When the rotor comes to rest, remove tubes and carefully aspirate the supernatant. Your lab
instructor will explain this procedure. The pellet now contains the cells. From your determination
of cell density calculate how many Tetrahymena are in the pellet. How many E. coli cells are in
the pellet?
To lyse the cells, suspend each cell pellet in 0.5 ml 0.1% SDS/1 mM EDTA. Break up the clumps
of cells using glass rods or vortex mixers. Immerse the tube in boiling water for a minute or two,
to help the proteins go into solution. How does SDS lyse cells and solubilize proteins?
Once the pellet is solubilized, dilute the sample 10-fold by bringing it to 5 ml with H
2
O. What now
is the concentration of SDS/EDTA? The protein of how many cells is in each ml?
4. Protein determination.
Refer back to the Bradford procedure of Part A. Prepare a new standard curve using BSA.
Compare this standard curve to the one prepared in exercise #1. Are they different? Do you see
why a new standard curve must be done each time an assay is performed? Serially dilute a 0.2
ml sample of each cell lysate 3 times, with 1.8 ml SDS/EDTA. You now have 4 tubes with
concentrations of cell lysate differing from one another by an order of magnitude. Transfer 1 ml of
each to new tubes. Add 4 ml of Bradford reagent to each. Measure the A595.
Calculate the amount of protein per tube. Correcting for dilutions, calculate the total amount of
protein per ml in each lysate. Knowing the number of cells that were collected to obtain each
lysate, calculate the amount of protein per cell for E. coli and for Tetrahymena. Express your
results in gms of protein per cell. Show all calculations.
Which cell has more protein? How do you account for this difference?
Suggestions for further study:

Protein content can be measured on extracts of other types of cells and by other procedures (see
Exercise II.A.). Cell growth can be monitored using this assay in parallel with other forms of
measurement (see Exercise I.D.). Factors influencing the rate of cell growth and the protein
content per cell can be evaluated.
Appendix: Spectrophotometry
An instrument that measures the amount of absorption (A), often referred to as optical density
(O.D.), of a colored solution is called a spectrophotometer. This instrument shines light of a
particular wavelength or frequency (monochromatic light) through the sample in a tube (called a
cuvette) to a phototube. The wavelength chosen is usually that which the particular color of the
solution absorbs most. The higher the concentration of a sample, the more intense is the color,
the more light is absorbed, and the higher is the optical density. This relationship is expressed by
the Lambert-Beer law:
Transmittance =I/I
o
=e
-kcl

where Io is the intensity of the incident light, I is the intensity of the light striking the phototube
after passing through a distance l (usually 1.0 cm) of solution of concentration c having a
characteristic absorption coefficient k, a constant. Optical density is usually measured in
absorbance units, because absorbance depends linearly on concentration. The relationship
between absorbance and transmittance is given in the following formula:
A =log (1/T) =log (I
o
/I) =kcl
Before measuring the absorption of a sample, the spectrophotometer must be set to an optical
density of zero with a blank, which is a solution containing all the ingredients of the assay, but
zero concentration of the molecule being assayed.
Spectronic 20 Operating Procedures
Identify the wavelength control knob on the top right, the amplified control on lower left front by
which the instrument may be adjusted to zero when no light is passing through it, and the light
control knob on the lower right front. Two scales are seen, the lower representing absorbance, or
optical density.
To operate the Spectronic 20, first turn the amplified control knob clockwise till it clicks. Let the
instrument warm up for about 10 minutes before using it.
Select the wavelength desired. With no cuvette in the instrument, and with the cuvette holder lid
closed, adjust the amplifier control until zero transmittance (or infinite absorbance) is indicated.
Insert the blank, and adjust the light control knob until the scale reads zero absorbance. Finally,
replace the blank with a sample and record the optical density.
For the instructor:
EQUIPMENT
Spectronic 20 or equivalent spectrophotometer and cuvettes (standard test tubes can be
substituted for cuvettes in a pinch, but do not give quite the same reproducibility)
Centrifuges to pellet the bacterial cells (high speed) and the Tetrahymena (clinical)
Hemocytometers
Compound light microscopes
SUPPLIES AND SOURCES
Cultures of Tetrahymena and E. coli and all standard biochemicals are available from biological
and scientific supply companies.
RECIPES
1. Luria Broth (for growth of E. coli)
5 gms NaCl
10 gms Bactopeptone
5 gms yeast extract
1 liter H2O
2. Enriched Proteose Peptone (for growth of Tetrahymena)
20 gms proteose peptone 5 gms dextrose 2 gms yeast extract
15 gms Agar 1 liter H2O
3. . Phosphate Buffered Saline
10 mM Phosphate Buffer pH 7.4 0.9% NaCl
The culturing tips from exercise I.D. (Cell culture) are reproduced here:
SUPPLIES AND SOURCES
Tetrahymena can be purchased as living cultures from most biological supply houses. They can
be maintained in 2% proteose peptone, passaging regularly. I have used sterile Erlenmyers (with
no more than 1-2 cm of medium in the bottom) or 100 mm sterile Petri dishes as culture vessels,
both quite successfully. To ensure vigorously growing cultures on the day of the exercise, it is a
good idea to transfer cells to a 10-fold excess of fresh medium the day before. Even allowing for
a lag before exponential growth resumes, this dilution allows for cells to be at or approaching their
maximum density at the time of the experiment. Then the 5-fold dilution into the culture media will
give countable numbers of cells (albeit high statistical variability) even at the earliest time points.
Proteose peptone and other microbiological media components can be purchased through Difco.
---2% proteose peptone: 1 g proteose peptone +distilled water to 50 ml. Dissolve with stirring.
Autoclave for 20 min. at 115
o
C (10 psi, less than the usual 121
o
C, 15 psi). Let cool. Store in the
cold. ---Complete medium (an alternative to proteose peptone, from Carolina's recipe as on insert
accompanying Tetrahymena cells): 5 g tryptone, 5 g proteose peptone, 0.2 g K2HPO4, 1 l water.
Adjust pH to 7.2. Autoclave. Store in the dark and cold until use.
Title: I. C. Cell Behavior
Author: J ason Wolfe, Biology Department, Wesleyan University
Purpose: To illustrate several complex cell behaviors in Tetrahymena and to introduce
approaches to analysis of their kinetics Time required: 2 to 3 hours
Level: Introductory
Introduction:
Some cellular processes are not easily reduced to individual biochemical reactions. Their
complexity may depend upon intact living cells. These processes may be studied at the cellular
level, rather than by disrupting cells and isolating their parts. However, the analysis can still be
quantitative.
Two "whole cell" activities will be quantitatively studied here: The kinetics of cell interaction and
the kinetics of phagocytosis. In addition, observations will be made on a third process, exocytosis.
1. Cell interactions
Tetrahymena not only eat by way of a "mouth" and portable "stomach" but they are also capable
of sex. When starved cells of two complementary mating types (there are seven mating types in
all) are mixed, they conjugate. Conjugation is a complex process involving a number of
cooperative interactions between individual mating types. First comes an invisible cell interaction
called costimulation, by which cells reciprocally trigger the onset of conjugation. Secondly, at
about an hour or so after costimulation cells attach to one another at their anterior ends to form V-
shaped doublets. The third interaction involves cytoplasmic coupling, and this occurs an hour
after pairing. Finally, gametic pronuclei are exchanged, fertilization takes place and a new genetic
complement replaces the old one.
2. Phagocytosis
Starved cells of the ciliated protozoan Tetrahymena will be used here. As this cell swims through
the medium it sweeps particles into its oral apparatus ("mouth"). The particles are then funneled
through a gullet into a sac which, when filled, pinches off into a food vacuole. The food vacuole
acts like a portable stomach. It circulates through the cytoplasm of the cell until its contents are
digested. Undigested residues are expelled through an anal pore.
3. Exocytosis
Bulk secretion occurs by the fusion of membrane bound vesicles to the plasma membrane.
Tetrahymena is a particularly good cell type with which to study exocytosis because it contains
about 5,000 large secretory vesicles per cell all primed for exocytosis. Secretion can be induced
such that exocytosis of the vesicles occurs synchronously within a cell and among cells in the
population. Finally, the product of secretion in Tetrahymena is directly visible. To assay for
secretion one can use the light microscope.
The secretory vesicle is called a mucocyst, because it contains a mucous-like substance. A
mucocyst is a torpedo-shaped structure about 1 um long containing mucus in a highly
condensed, paracrystalline state. The polyanionic dye, Alcian blue, triggers exocytosis causing
the contents to be released from the cell, whereupon the mucus absorbs water and swells into a
gel. When mucocysts are secreted synchronously the separate gels coalesce into a visible
capsule which surrounds the cell. The cell can rotate within the capsule, or can break out of it,
leaving an empty gelatinous chamber behind. Interestingly, Alcian blue also stains the capsule
(because of its sulfated mucopolysaccharide content) so the capsule can be seen with a light
microscope using bright light or phase contrast.
Procedure:
1. Kinetics of Cell Pairing:
Because sex in Tetrahymena takes longer than eating, this exercise will start with the kinetics of
conjugation. Two mating types have been starved overnight in dilute buffer (10 mM Tris, pH 7.4)
in preparation for conjugation. Before class begins, equal numbers of cells of both mating types
(BIII and BVII) will be mixed together and incubated at 30xC. The cell density should be
approximately 5 X 10
5
cells/ml. For effective conjugation the surface-area- to-volume ratio is kept
fairly high. As a rule, the cells should be in a flask 10 times the volume of cell suspension.
At hourly intervals transfer a drop of cells to a microscope slide and examine by phase contrast
microscopy. Make observations on the behavior of the living cells as they form cell pairs. Record
your observations in your notebook. At the same time intervals, take 0.5 ml from the flask,
transfer it to a disposable test tube, and fix the sample by adding an equal volume of buffered
20% formaldehyde* in 0.01 M phosphate buffer at pH 7.0.
*CAUTION: Do not mouth pipette formaldehyde. Approximate 0.5 ml with a disposable plastic
Pasteur pipette. Alternatively use a less volatile fixative, such as glutaraldehyde.
To assay for the percentage of cells in pairs, transfer a drop of cells to a microscope slide and
add a cover slip. Using the 10X phase objective, scan the slide methodically from one edge of the
coverslip to another. As you encounter a single cell, call out "1" and when you encounter a
conjugating pair call out "2". Your partner will record these numbers in 2 columns, one for single
cells and one for conjugant pairs. Stop counting after 200 cells have been encountered. Total up
the numbers and determine the percentage of conjugation as follows:

#of cells in pairs
% conjugation =100 x ----------------------------------------

#of single cells +#of cells in pairs
On a piece of graph paper plot the percentage of conjugation (Y axis) vs. time in hours (X axis).
When did cell pairing first begin? What is the plateau value for conjugation? What is the shape of
the curve? What would you expect the shape of the curve to be if cell pairing were a function of
random collisions between selectively "sticky" mating types? What conclusion can you draw
about the conjugation process from your kinetics?
2. Kinetics of phagocytosis
After taking your 60-minute time point for conjugation, you have a full hour for a study of
phagocytosis. To monitor the uptake of food particles into food vacuoles, we will substitute food
with opaque inert particles. A highly suitable material is India ink, which is made up of a
suspension of minute black particles. When taken into a food vacuole, they are compressed
together into a black ball.
Transfer 0.5 ml of starved Tetrahymena cells into 4 disposable vials. Add to each 0.5 ml of 1.0%
(v/v) India ink in Tris buffer. After 2, 5, 10, and 20 minutes fix each with 1 ml formaldehyde, as
above.
Examine the cells in the microscope using bright field optics. Scan the slide as above, and score
the cells for kinetics of food vacuole formation. Set up columns labeled 0-15. As you encounter
each cell call out the number of food vacuoles (black balls) you count. Let your partner record this
in the appropriate column. Make a note of the variation in size and shape of food vacuoles. Do
you see any dividing cells? Do they form food vacuoles?
Total up the number of cells in each column. On a piece of graph paper plot the percentage of
cells with food vacuoles (Y axis) vs. time in minutes (X axis). On a separate piece of graph paper,
held vertically, draw 4 histograms, one for each time point, illustrating the percentage of cells with
0, 1, 2, 3...n, food vacuoles.
From the two sets of data can you estimate how long it takes, on the average, for a cell to form a
food vacuole?
3. Exocytosis
Obtain 9.5 mls of cells at a density of ~10
5
cells/ml that have been starved in Tris for 1 1/2 - 2
hours. Add 0.5 ml of 1% Alcian blue in 0.05 M sodium acetate. Immediately place a drop on a
slide and observe blue capsules by both bright light and phase contrast microscopy. Find cells
rotating in capsules, or trying to swim out of them. OPTIONAL - It is sometimes thought that
calcium plays a role in secretion. Repeat this experiment but first equilibrate the cells in Tris with
EGTA (0.1 - 5 mM) for 5 min. EGTA binds free Ca
++
making it inaccessible to the cells.
Observe cells after adding Alcian blue. Do you see capsules? Explain your results. Record your
observations. Suggestions for further study:
4. Competition between eating and sex
A. Incubate cells of each mating type in India ink for 10 minutes, and then mix
together. Incubate for 2 hours at 30xC, then fix a sample and score for the
percentage of cell pairing. Compare this value to a 2 hour control sample. (A
rigorous control would have to take account of the dilution created with the India
ink under conditions in which there were no India ink). Has feeding behavior
altered sexual behavior? If so, how?
B. Withdraw small samples (e.g. 0.5 ml) of cells from a conjugation mixture at 1,
2 or 3 hours after mixing mating types. Add to an equal volume of India ink and
incubate for 5 minutes. At the end of this interval, fix the sample. Score for the
number of food vacuoles in single cells and in paired cells. Has sexual behavior
altered feeding behavior? If so, how?

For the instructor:
EQUIPMENT
SUPPLIES AND SOURCES
RECIPES
COMMON PROBLEMS AND THEIR LIKELY CAUSE ? What is the pH of the sodium acetate ?
Are dividing cells readily distinguishable? or should the students give the criteria they use in
identifying them as dividing?
Title: I.D. Cell culture
Author: Mary Lee S. Ledbetter, Department of Biology, College of the Holy Cross
Purpose: To understand the conditions that limit cells' ability to survive and proliferate in vitro.
Time required: 3 hours of lab time +several 15-minute visits to take time points over the next 24
hours. Since the carrot cell culture takes a long time, it is best scheduled early in the semester.
Level: Introductory
References: L.P. Everhart (1972) "Methods with Tetrahymena" in D.M. Prescott, ed., Meth. Cell
Physiol. 5: 219-287.
Introduction:
Techniques for growing cells in the laboratory were originally developed by microbiologists. The
rapid generation time of most microorganisms as well as their autonomous growth and relatively
few nutritional requirements made it easy to provide the cells with an optimum environment and
to avoid contamination by other organisms. In the past 50 years these techniques have been
extended to the culture of cells from higher plants and animals. Using cell culture, one can
address questions relating not only to cell growth per se but also to nutritional requirements,
metabolic activity, gene expression, motility, and features of differentiation, aging, and regulation.
The chief advantage of animal or plant cell culture is the control the investigator can exert over
the cells' environment in the absence of influences from other systems of the multicellular
organism. It is important to keep in mind, however, that the laboratory culture cannot reproduce
every detail of the cell's natural surroundings, and some of the missing properties may be
significant to the process under study. Thus cell culture is a model system, a simplified version of
what is understood to be "real life."
Today we will explore two kinds of eukaryotic cells in culture: the protozoan Tetrahymena
pyriformis and the dicotyledonous plant Daucus carota (carrot). The former is a single-celled
organism, naturally free-living in most freshwater ponds, where it survives by ingesting bacteria
and other particles into its food vacuole and digesting them. This environment is not too difficult to
mimic in culture. The carrot, in contrast, is a complex, multicellular organism whose various
tissues are composed of interdependent, differentiated cells. This environment is more difficult to
duplicate in the laboratory. But the differentiated state in most nucleated plant cells is not
permanent. We can thus culture a piece of differentiated tissue (or even a single cell) on the
appropriate nutrient medium, and what grows is an undifferentiated mass of cells called callus.
Transferring callus to an environment with the appropriate plant hormones induces differentiation
of roots, shoots, and ultimately a new plant.
Tetrahymena cells have complex nutritional requirements, including essential amino acids,
vitamins and minerals as well as the sources of nitrogen and carbon needed by all organisms. All
these requirements may be provided by proteose peptone, a commercially available partial
hydrolysate of protein. We will grow our Tetrahymena in a medium consisting of 2% proteose
peptone in water, autoclaved for sterility.
When growth is not limited by the composition of the medium, cells are said to be in "exponential
phase" or "logarithmic" growth: in each successive time period the size of the population
increases by a constant factor. Represented mathematically,
dN

----- = kN,
dt


where N is the number of cells present at time t and k is a growth constant that depends on that
cell type and those growth conditions.
Rearranging: dN


----- = kdt

N


Integrating between (N
o
,t
o
) and (N,t): ln (N/N
o
) =kt
The population doubling time is the value of t for which N/No =2. Moreover, a plot of ln (N/No) as
a function of t will be a straight line whose slope is k. (Even simpler is to use semilogarithmic
graph paper, plotting t along the evenly spaced axis and N/No on the axis perpendicular to it.) If
you know the initial number of cells and the growth rate constant, the size of a culture in log
phase can be predicted at any subsequent time.
Tetrahymena growing at their fastest rate divide once every 3 to 4 hours. It wouldn't take long at
that rate for them to outweigh the earth! Fortunately logarithmic growth is usually limited by the
availability of nutrients or the environment's carrying capacity for waste. At that point the
population growth slows until the culture is in stationary phase, where cell death balances cell
division and even cell division may be slowed. Cells in adult organs of most higher animals are in
stationary phase. If conditions become sufficiently adverse, a death phase may ensue. A growth
curve is the result of an experiment to measure the characteristics of the various phases of a
population of a certain cell type under a defined set of conditions.
When working with either Tetrahymena or carrots, it is absolutely essential to maintain sterility.
Even the 3-hour generation time of Tetrahymena pales in the face of typical bacterial
contaminants that double in number every 30 minutes, and the 1- to 2-hour doubling time of
molds. Carrot cells divide even more slowly. Several measures are customarily taken to ensure
sterility:
1. Sterilize everything likely to come into contact with the culture. Media and glassware may be
autoclaved; disposable plasticware has been sterilized by radiation or treatment with gas, and
should remain wrapped before use; tools and implements can be dipped in alcohol and flamed.
2. Work in a clean protected area whenever possible. The ideal situation is to use a laminar flow
hood, whose air environment is filtered to remove particles. Alternatively you can use a protective
box covering your work area. If it is necessary to work on the open bench, take precautions to
keep dust levels down.
3. Avoid opening sterile containers for longer than necessary to sample or inoculate.
4. In some cases it is advisable to include antibiotics such as penicillin or streptomycin in the
culture medium.
Key features of sterile technique will be demonstrated at the beginning of lab.
Procedure
Part One. Growth properties of a population of Tetrahymena pyriformis and the dependence of
growth rate and saturation density on composition of the medium and temperature.
A stock culture in late log phase will be provided in a 30
o
water bath. Each group will prepare two
growth curves on these cells, one under optimum conditions (30
o
C, 2% proteose peptone) and
one in which one of these variables has been altered (lower or higher temperature OR 0.4%
proteose peptone). The suboptimal conditions will be assigned at random.
Using sterile technique, transfer 4 ml of the stock culture to a sterile Erlenmyer flask (labeled A
and group's initials) containing 16 ml of 2% proteose peptone. Mix the culture thoroughly. Initiate
a second culture with another 4 ml of the stock as directed. Label with B and the group's initials.
Sterilely sample 0.5 ml of A to a small test tube containing 0.1 ml 3% glutaraldehyde. Note time.
Place culture A in the 30
o
water bath. Sample culture B in the same way. Note time. Place culture
B in its appropriate incubation environment. These times are the starting points for the two
cultures, and elapsed time should be calculated relative to them.
Determine the concentration of cells in the two glutaraldehyde-fixed suspensions using the
hemocytometer. Once the cells are fixed, they no longer need be handled sterilely.
At intervals of 20 to 30 minutes repeat the sampling of your two cultures, carefully noting the time.
The fixed samples can be counted as you go, or they can be saved for several days if necessary.
In addition to samples taken during the regular laboratory period, you should make provisions for
samples to be taken several times during the evening and the next morning. (Assign your
volunteers before you leave!) Pay close attention to sterile technique when sampling.
Cell counting with the hemocytometer
Be sure the hemocytometer chamber and its special coverglass are clean and dry. Place the
coverglass over the chamber. Using a non-sterile Pasteur pipette, suspend the fixed cells and
sample about 1/3 of the volume. Discard the first two drops. Allow the next volume to flow by
capillary action into one side of the chamber under the coverslip until it is filled; then fill the other
side. Do not let the chamber overflow. Place the chamber under the low power objective on your
microscope. Count all the cells in each of the 4 corner squares and the central square on one
side. Repeat for the other side. Include in your count cells that lie on the left and top borders of a
square; ignore those that lie on the right and bottom.
Calculate the mean and standard deviation of your 10 readings, the average number of
cells/square. The concentration of cells in the sample is given by that mean count x 10
4
, in units
of cells/ml. Correct this concentration for the dilution introduced by the fixative to get the cells/ml
in your original culture. Provide these data to the instructor by Friday. On Monday you will receive
a copy of the data from all the groups to analyze for your reports.
If you have calibrated the fields of view of your microscope, you can verify that the volume over a
square corresponds to the conversion factor given in the previous paragraph. The distance from
the slide to the coverslip of a hemocytometer is 0.1 mm. Measure the dimensions of a square
using your knowledge of the size of the 10x field of view. Calculate the volume over the square; it
should be 0.0001 ml. (Remember: 1 ml is about 1 cm
3
.)
During waiting periods between time points, try two other methods for estimating cell
concentration:
1) Turbidity, measured as apparent absorbance at 450 nm in the
spectrophotometer. Each particle in the suspension scatters a unit of light, so the
light scattering should be proportional to the number of particles. This procedure
is useful if the particles do not settle too rapidly. Sterilely pour a sample of cells
from the large culture in the water bath into the Spectronic cuvette. Read its
"absorbance" compared to a blank solution containing sterile medium. Note the
time and your reading on the data sheet next to the spectrophotometer. Discard
the solution when you have finished. If everyone participates, we can get a
growth curve from the group results.
2) Electronic cell counting, in which a particle passing over a charged orifice interrupts the charge
and is recorded as a pulse. The number of pulses is a measure of the number of particles in the
volume that flowed past the orifice. This procedure is useful if you have large numbers of cells
and many samples to count, as in a clinical hematology laboratory. If an appropriate instrument is
available, dilute 0.1 ml of one of your fixed samples into 20.0 ml isotonic saline in a special plastic
vial. The instructor will help you use the Coulter (or other) counter to measure the cell
concentration. Compare the value you obtain with the concentration for that same sample in the
hemocytometer.
Part Two Initiation of a carrot cell culture from a sample of carrot root. (adapted from the
procedure described in the instructions accompanying the kit from Carolina Biological Supply Co.,
1982)
This experiment is long range. We will initiate cultures today and examine them periodically over
the course of the semester for the development of callus. We will prepare "explants," small pieces
of tissue, from carrot roots and place them on callus initiation medium, a salt base supplemented
with sucrose (a carbon source); the vitamins thiamine (B
1
), pyridoxine (B
6
), and nicotinic acid (B
3
),
normally provided by other parts of the plant; the synthetic auxin (plant hormone) 2,4-
dichlorophenoxyacetic acid, more light-resistant than the natural counterpart, indoleacetic acid,
which stimulates cell division; and agar to provide a semi- solid support. Once callus has formed,
pieces can be subcultured on shoot development medium, similar to callus initiation medium but
lacking the auxin.
A sterile work area has been set aside. Keep it clean and wipe it just before use with 70%
ethanol. Stand or sit back from the work area, inserting only your hands and your sterile tools. In
particular avoid loose items of clothing or hair entering the area, and try not to breathe on your
sterile items. Wash hands with soap and 70% ethanol. Before you begin, make sure you have
everything you need within reach and that tops of containers have been loosened to permit easy
opening. These include forceps, scalpels, ethanol, sterile distilled water, paper towels, a
wastebasket, and a container to hold discarded solutions.
Sterile instruments are kept in a jar of ethanol until they are needed. To remove the ethanol, dip
the instrument into the sterile water and proceed. When finished with that instrument, wipe any
debris on a paper towel and return the instrument to the ethanol. Be sure that "dirty" operations
are not performed over "clean" or sterile items.
PREPARATION OF ROOT EXPLANTS
The instructor will have prepared pieces of carrot taproot before lab. Carrots were gently washed
in warm soapy water. Working in the sterile work area, the instructor broke off and discarded the
ends of the carrot. A sterile cork borer was then used to remove a core from the region between
these newly exposed ends outside the central core of the root. Alternatively, rootlets of newly
germinated carrot seeds can be used. Each student or group is given a sterile Petri dish with one
carrot core (or the rootlets from several seeds) and several dishes of callus initiation medium.
When it is your turn to prepare your explant, resterilize the work area with ethanol. Check to be
sure all your needed supplies are at hand.
1. Dip forceps and scalpel in ethanol and then sterile water. Open the dish containing the core.
Cut off about 1 cm at both ends of the core and discard the ends (or use the entire rootlet). Cut
the remaining piece of core into 2-to 4-mm thick cross sections. Replace the lid on the dish. Wipe
any debris from your tools and return them to the ethanol.
2. Remove forceps from ethanol, and dip them into sterile water. Take a dish of callus initiation
medium and carefully lift the lid. Using the forceps, lay two or three cross sections of carrot core
on the agar. Close the dish and mark with the tissue source, date, and your name. Repeat with
the other dishes until your core sample is completely distributed. If you are using rootlets, try
leaving some intact while cutting the others into a tip end and a seed end.
3. Seal the dishes with tape or Parafilm or put them into a clear plastic bag to keep them from
drying out. Place the cultures in indirect light at a constant temperature of 24
o
to 27
o
C. The ideal
light/dark cycle is 16 hr/8 hr. Clean up the sterile work area for the next students.
4. In a few days check your dishes for contamination. If it appears, remove the affected dish at
once. Do not open the dish; tape it and discard it immediately to prevent spread of the
contamination.
In one to two weeks, callus should begin to form on the explants. In four to six weeks, the callus
should be 1 cm in diameter.
TRANSFERRING THE CALLUS
Four to six weeks after explant initiation, the callus is transferred to shoot development medium to
induce shoot and root formation. Remove your dishes of callus tissue from the incubator and take
them to the sterile work area. Check to be sure you have all the necessary supplies.
1. Remove the tape from each dish and dip your forceps in ethanol and then in sterile water.
Open each dish and transfer the callus to a dish of shoot development medium. Pieces of callus
larger than 1 cm in diameter can be divided and used to replace cultures lost from contamination.
Label the dishes with the date and your name.
2. Seal the dishes with tape and place in indirect light. Clean the work area. Check the dishes in a
few days for contamination.
3. In three to four weeks, check the callus through the lid of the dish with a hand lens or a
stereomicroscope for signs of shoot and root formation. Turn the dishes over and look for roots
growing into the agar. You may find areas where a few cells were broken away from the callus
clumps; look for signs of growth and organization in these.
4. In six to eight weeks, the callus should be producing roots and shoots big enough to see with
the naked eye. When the shoots are about 3 to 5 cm long, the plantlets may be transplanted.
TRANSPLANTING (Optional: usually the semester has ended by this point) Sterility is no longer
necessary at this stage.
Add about 1 liter of water to 4 liters of any soilless potting medium and mix well. Allow the
moistened medium to stand overnight. Mix the potting medium again before using. Small pots or
cups with holes in the bottom can be used as containers. Place a tray under the pots and loosely
fill the pots with potting medium; do not pack.
Select plantlets that are at least 3 cm tall. There may be several plantlets per callus piece, and
these can be cut apart with a scalpel. Bring the plantlets from the sterile work area to a sink.
Rinse under lukewarm running water until all medium that was attached to the plantlet is
removed. Otherwise residual medium can support the growth of bacteria and fungi that may kill
the tender plantlet.
Place one plantlet in each pot and gently press potting medium around it. Keep moist by adding
water to the tray and allowing plants to soak up water from the bottom. About once a week, water
with one-fourth strength liquid fertilizer containing equal portions of nitrogen, phosphate, and
potash. The first week it may be necessary to cover the pots with a piece of clear plastic or plastic
wrap. This can gradually be removed as the plants become hardened off, or acclimated to their
new surroundings.
Suggestions for further study:
Similar approaches can be used to culture callus from the leaves of African violets. A kit is
available for this (VioClone), or one could experiment with the methods described above. In this
case it is important to remove surface contamination by dipping the leaf in soapy water before
cutting it into segments with a scalpel. The results can be compared with those obtained by taking
a leaf cutting and immersing it in water in a clear test tube.
A faster growing eukaryotic microorganism is yeast; judicious choice of mutant yeast can reveal
interesting features of nutrient metabolism.
For the instructor:
EQUIPMENT
Part One:
Water bath(s)
Autoclave (for preparation of media and glassware)
Hemocytometer and coverslip
Compound light microscope.
Part Two:
An incubator or environmental room where cultures can be maintained at 24
o
to 27
o
C. Ideally,
the photoperiod should be 16 hours of light and 8 hours of darkness. Use bright indirect natural
light or fluorescent plant growth lights, never direct sunlight, as this will probably kill the cultures.
A sterile work area: If it is available, use a horizontal laminar flow hood unit. Alternatives include a
transfer case or a corrugated paper box or aquarium set on its side so the opened top faces the
culturer. Keep the work area clean in a draft-free area and wipe it just before use with 70%
ethanol.
SUPPLIES AND SOURCES
Tetrahymena can be purchased as living cultures from most biological supply houses. They can
be maintained in 2% proteose peptone, passaging regularly. I have used sterile Erlenmyers (with
no more than 1-2 cm of medium in the bottom) or 100 mm sterile Petri dishes as culture vessels,
both quite successfully. To ensure vigorously growing cultures on the day of the exercise, it is a
good idea to transfer cells to a 10-fold excess of fresh medium the day before. Even allowing for
a lag before exponential growth resumes, this dilution allows for cells to be at or approaching their
maximum density at the time of the experiment. Then the 5-fold dilution into the culture media will
give countable numbers of cells (albeit high statistical variability) even at the earliest time points.
Carrots should be obtained from a natural food store or from your garden, since my experience
has been that supermarket carrots seem to grow less well, perhaps due to their having been
treated with preservatives.
Proteose peptone and other microbiological media components can be purchased through Difco.
Callus initiation medium and shoot development medium is available from Carolina Biological
Supply or can be prepared on site (see below).
RECIPES
Part One
---2% proteose peptone: 1 g proteose peptone +distilled water to 50 ml. Dissolve with stirring.
Autoclave for 20 min. at 115
o
C (10 psi, less than the usual 121
o
C, 15 psi). Let cool. Store in the
cold.
---Complete medium (an alternative to proteose peptone, from Carolina's recipe as on insert
accompanying Tetrahymena cells): 5 g tryptone, 5 g proteose peptone, 0.2 g K2HPO4, 1 l water.
Adjust pH to 7.2. Autoclave. Store in the dark and cold until use.
---0.1% proteose peptone: 1 part sterile 2% proteose peptone, 19 parts sterile distilled water. Mix
in a sterile container.
Part Two
---Gamborg's B-5 medium (ref. Gamborg, Miller and Ojima, Exp Cell Res 50:151 (1968)). This
can be purchased as a dry powder from GIBCO; if kept in the refrigerator, it has a shelf life of at
least a year. It can then be prepared according to the package directions, though I have
personally used only the pre-made agar medium supplied with the Carolina kit. GIBCO also sells
2,4-D and phytagar. The preparation is as follows:
1. Sprinkle the desired amount of powdered medium into 20% less distilled deionized water than
ultimately needed, with stirring. Mix until completely dissolved.
2. Add 2 mg/l 2,4-D to half of the batch (designated callus initiation medium); the remaining half is
shoot development medium.
3. Adjust pH to 5.7, if necessary, with 1 N KOH or 1 N HCl.
4. Add 8 g/l of agar (GIBCO's tissue culture grade Phytagar comes highly recommended).
5. Dilute to final volume with distilled water. Heat gently until the solution clears, indicating the
agar has dissolved.
6. Autoclave 15 min (or longer for >200 ml) at 15 psi (=121
o
C); slow exhaust.
7. Cool until you can handle the flask without burning yourself, but don't let the agar gel. Pour the
plates, several mm thick. Mark the ones that have the 2,4-D. Let gel, then refrigerate in a closed
container to prevent dessication.
Preparation of carrot cores: Needed are carrot taproots from a garden or health-food store, a jar
of ethanol, a cork borer, enough sterile 100x10mm petri dishes for students in that day's class,
and wooden applicator sticks. Begin with all but the carrots at the sterile work area.
1. Wash carrots in warm soapy water, but do not scrub. Remove any remaining leaves and stem.
2. Take the roots to the sterile work area to proceed. Use sterile technique for the remaining part
of this procedure.
3. Place the cork borer and applicator sticks in a jar of ethanol. Remove the petri dishes from the
package.
4. Holding the carrot in your hand, first break off the large end, then the small end, and discard
these ends. Be careful not to touch the newly exposed surfaces. If the center section is longer
than the cork borer, break this section in half so each section is no more than 7 cm long.
5. Put the root, large end down, in a sterile petri dish into which a little ethanol has been poured.
Remove the cork borer from ethanol. While holding the carrot in place, push the cork borer into
the region outside the central core of the root. Be sure the borer goes all the way through the root
until it is in contact with the plate. Turn the borer several times while gently pressing down to be
certain the core is free.
6. Pull the cork borer out. Take the applicator stick out of ethanol and insert it into the handle end
of the cork borer. Open a sterile petri dish and gently push out the core into the dish; close the lid
and set aside. Use a separate dish for each core. Repeat until you have a core in each dish.
Preparation of carrot rootlets:
1. Soak carrot seeds overnight to accelerate germination, then rinse briefly in a dilute detergent
solution and spread on sterile filter paper moistened with sterile water in sterile petri dishes. Store
in the dark where they won't dry out.
2. When the primary rootlet is ~2 cm long, the material is ready to use. To prepare for class, use
a sterile forceps to grasp each rootlet, dip it in a dilute solution of detergent in sterile water and
place it in a sterile petri dish.
3. With a sterile scalpel cut off the seed end of the rootlet; transfer the rest to a second petri dish.
You can use the same petri dish for several rootlets.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
Part One
Tetrahymena grow relatively slowly and saturate most media at no more than 2 to 3 x 10
5
cells/ml
(20-30 cells/square in a hemocytometer). Thus occasionally the earliest time points made from
diluting the original stock culture 5-fold into the various growth media show a high degree of
variability, since students are trying to calculate averages of numbers that vary around 4-6
cells/square. Not surprisingly, they will often see an apparent decline in cell number in the first
hour of the experiment, but usually if they are asked to consider the variance of the numbers,
they will realize that they cannot distinguish any change in that time period. Often measurable
growth can be detected by the end of the period, but the real growth is usually seen in the
evening and next morning. Depending on how eager the students are to collect time points, they
can easily discriminate among the various treatments.
Since Tetrahymena in the wild eat bacteria, contamination is less of a problem than with many
other kinds of cell culture. Still I have in the past encountered contaminants that suppressed the
protozoan growth.
The least successful measurement in this exercise has been the turbidity measurement, for
reasons not entirely clear to me. If anyone has an old-style nephelometer and Erlenmyer flasks
with side arms, they might be preferable to the Spectronic 20 as measuring devices. If a side-arm
flask is used with a Spec 20, the instrument should be used in a darkened room, since stray light
significantly interferes with readings.
Part Two Contamination is the principal problem with this exercise. When working with root cores,
it is easy to slip and touch the material with your fingers. When working with rootlets, they can
carry over mold contaminants even if germinated as carefully as possible. Mold becomes visible
within a very few days. Even if it appears to involve only one of a group of explants in a dish, the
entire dish should be discarded.
A second problem is dessication. If the agar medium dries out, so will the explants and you will
get no growth. If you can humidify the area where the cultures are incubating, that is best;
alternatively they can be placed in plastic bags with damp towels.
The worst aspect of the exercise is the time it takes to see whether it has worked. For that
reason, I generally do it as an adjunct to some other, more immediately productive, activity, as
described here.

Title: II. A. Purification of mitochondria by differential sedimentation and monitoring of fractions for
specific activity of succinate dehydrogenase
Author: Mary Lee S. Ledbetter, Department of Biology, College of the Holy Cross, Worcester,
Massachusetts 01610
Purpose: To integrate microscopic observation and quantitative analysis with cell fractionation
Time required: variable. Well prepared students can accomplish the entire laboratory in a single
4-hour period, but I ordinarily use 2 or even 3 3-hour periods to ensure mastery.
Level: Intermediate
References: Cell fractionation is described in most comprehensive cell biology textbooks.
Biochemical assays for protein and for enzyme are also widely discussed.
The procedure below is adapted from one developed at Dartmouth College by J ames Yager.
Part A: Preparation of cell fractions Introduction:
Eukaryotic cells contain many kinds of subcellular organelles. In order to determine the physical
and chemical properties of these organelles, it is necessary to study them in isolation from other
cellular components. The most common way to perform this cell fractionation is disrupt the cells
and separate their components by use of centrifugation. Particles subjected to the acceleration of
a centrifugal field will move, or sediment, at a velocity determined by various properties of the
particle, including its mass, its density compared to that of the suspending fluid, and its frictional
properties.
If the suspending fluid forms a gradient of density around the density of the particle itself, the
particle will form a band at a position in the centrifuge tube where its density is matched by the
density of the fluid. The forces impelling it further down the tube are countered by the increasing
resistance of the ever denser fluid. The tendency of diffusion to disperse the particles is
countered by the centrifugal field. Cell homogenates treated in this way can be separated in one
step into multiple bands of species differing in density. This technique is known as equilibrium
density gradient centrifugation. The best separation, however, occurs only after centrifugation for
a long time, when equilibrium has become established between the forces concentrating the
particles and those leading to their dispersion.
A less complete, but more rapid, separation can be achieved by a technique called differential
sedimentation. The fluid in which the particles are suspended is less dense than any of the
particles. Thus instead of forming bands, they pass down the centrifuge tube at a rate determined
by the properties described above. When they reach the bottom of the tube, they form a pellet. If
particles of several different kinds are present initially, the pellet's composition will change over
the period of the centrifugation, initially consisting of the most rapidly sedimenting particles, with
the slower ones being added later. Thus a separation can be effected by stopping the
centrifugation periodically, removing the pellet, and continuing to centrifuge the supernatant.
This procedure has been standardized into a scheme for fractionating cells, in which the various
pellets have been identified according to the predominant organelle they contain: the nuclear
fraction, the mitochondrial (or plastid) fraction, the microsomal fraction, and the soluble fraction. It
is important to remember, though, that each fraction is contaminated by elements of the other
fractions, unless further purified. Also the soluble fraction contains material, e.g. ribosomes, which
could be pelleted if the centrifugal forces were further increased. Thus, in a rigorous application, it
is necessary to confirm by independent means the purity of a fraction. This can be done by
microscopic analysis or by assaying for an enzyme activity characteristically associated with a
particular organelle.
The rate at which a particle moves is also a property of the centrifugal field, usually expressed as
a multiple of g, the acceleration due to gravity. Thus the relative centrifugal force, R.C.F. =1.119
x 10
-5
(rpm
2
)r, where rpm is the revolutions per minute of the rotor and r is the distance (in cm) of
the particle from the axis of rotation. A convenient way to determine the R.C.F. for a given rotor at
a given speed is by use of a nomogram (see Figure II.A.1). The radius used is usually the mean
between the radius to the top of the centrifuge tube and the radius to the bottom.
Since distance =rate x time, a given particle may be sedimented the length of the centrifuge tube
(i.e., pelleted), by subjecting it to high speeds for short times or to lower speeds for longer times.
Other factors influence the choice of conditions, such as diffusion properties of the particle of
interest compared to other particles in the homogenate, or other time-dependent factors.
Various kinds of centrifuge are available to carry out this kind of work. Clinical centrifuges, both
refrigerated and not, seldom run faster than 5,000 rpm; they can readily sediment cells, nuclei, or
chloroplasts, but smaller particles require impractically long times. High-speed centrifuges go as
fast as 18,000 rpm; ultracentrifuges can manage 60,000- 75,000 rpm. Both these types of
instrument are usually refrigerated and may also be operated in a vacuum, to reduce heat
generated by friction with air molecules. The rotors, too, are designed of titanium and strong
alloys to withstand the strain of the centrifugal forces and to conserve angular momentum.
Procedure:
The exercise today will use differential sedimentation to prepare three fractions from a
homogenate of rat liver: a "nuclear" fraction, a "mitochondrial" fraction, and a "soluble" fraction
(which nevertheless still includes cell components such as microsomes and ribosomes, that could
be sedimented in stronger centrifugal fields).
1. (Done before lab. Come early if you want to see). Kill the rat by overanaesthetizing with ether
or guillotining. Open the abdomen and perfuse the liver with ice-cold buffered sucrose* to remove
excess blood and rapidly chill the cells, preserving enzyme activity. Excise the liver to cold
buffered sucrose on ice. Trim away fat and connective tissue.
*Buffered sucrose: 0.25 M sucrose in 10 mM phosphate buffer, pH 7.0.
2. Place 20 ml ice-cold buffered sucrose in a beaker on ice. Weigh. Add about 2 g fresh- perfused
liver. Reweigh. Calculate exact weight of liver. Mince the tissue fine with scissors. Rinse the
minced tissue several times with fresh, ice- cold sucrose. After the last rinse, measure out a
volume of sucrose equal to 9 times the tissue weight. Add half to the minced tissue. Save the rest
on ice.
3. Pour tissue pieces to a homogenizer vessel. Homogenize 15 times, until no lumps remain.
Keep cold throughout. Decant through cheesecloth into an ice-cold centrifuge tube. Rinse the
homogenizer vessel with the reserve sucrose solution and pour it through the cheesecloth into
the tube too. This is a 10% (w/v) homogenate. Estimate and note the volume. Save a small
measured amount (2 ml or so) in a chilled test tube for assays and microscopy.
4. Centrifuge 10 min at 600 x g at 4
o
C. (1,600 rpm in the IEC centrifuge using the swinging bucket
rotor #269, r =19.8 cm).
5. Pipette the supernatant to a fresh, ice-cold centrifuge tube. Note the volume. Resuspend the
pellet in 20 ml buffered sucrose. Centrifuge again for 10 min at 600 x g to wash the nuclei.
Discard the supernatant. Resuspend the pellet in 5 ml buffered sucrose. This is the nuclear
fraction. Save it on ice.
6. Centrifuge the supernatant (from step 5) for 20 min at 10,000 x g (8,600 rpm in the Beckman
J 2-21 centrifuge using the #J 17 fixed-angle rotor, r =12.3 cm).
7. Pipette the supernatant to a fresh, ice-cold test tube. Note the volume. This is the soluble
fraction. Resuspend the pellet in 5 ml ice-cold sucrose. This is the mitochondrial fraction.
8. You now have four samples: the crude homogenate and the three fractions.
a. Prepare wet mounts of each and examine by phase microscopy. Record
observations of the kinds of particles present and their relative frequencies.
b. Prepare slides stained with methyl green pyronin:
--Place a drop of the fraction on a clean slide.
--Spread the drop with the edge of a second slide to make a smear.
--Allow the slide to air dry with gentle heat.
--Add 3-4 drops of stain. Let sit 3 minutes.
--Immerse slide in a rack in a pan of running tap water for about 5 min.
--Add 1 drop glycerin and a coverslip.
--Observe in bright field. Nuclei should stain green, cytoplasm red or pink, and
mitochondria can be seen as small dots.
9. If this is to be a stopping point, divide each fraction equally between two test tubes. Freeze one
for the protein determination and the other for the enzyme assay. Label the tubes with your initials
and the fraction designation.
Part B: Protein determination Reference: Lowry, O.H., N.J . Rosebrough, A.L. Farr and R.J .
Randall (1951) Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193: 265- 275.
Introduction:
The paper describing this assay procedure is one of the most widely referred to in the biological
literature. It permits the determination of protein in amounts as dilute as 10 ug/ml by measuring
the intensity of the blue color that develops after first reacting the protein solution with alkaline
copper and then with Folin's phenol reagent. The Cu
++
ions form a pink-to-violet chelation
complex with peptide bonds of proteins in alkaline solution. The phenol reagent is a
phosphomolybdic-phosphotungstic acid reagent which is reduced to a dye, molybdenum blue, by
aromatic amino acids, such as tryptophan and tyrosine. The action of both reagents leads to a
stable and sensitive color whose maximum absorbance is at 650 nm. Procedure: 1. Preparation
of samples: Thaw one sample of each fraction from last week. Dilute your fractions in buffered
sucrose as follows:
homogenate 1/10, 1/20
nuclear 1/5, 1/10
mitochondrial 1/5, 1/10
soluble 1/5, 1/10
For each dilution sample 0.1 ml into each of 2 clean 13 x 100 tubes. Add 0.9 ml distilled water
(another 1/10 dilution).
2. Prepare a standard curve and blank. To duplicate 13 x 100 tubes add
0.1 ml BSA +0.9 ml H2O
0.2 ml BSA +0.8 ml H2O
0.3 ml BSA +0.7 ml H2O
0.4 ml BSA +0.6 ml H2O
0.5 ml BSA +0.5 ml H2O
1.0 ml H2O
BSA refers to bovine serum albumin, a protein used as a reference standard and provided to you
at a concentration of 200 ug/ml.
3. Prepare reagent C: In a beaker, mix 200 ml reagent A (20 g/l Na2CO3, 4 g/l NaOH, 0.2 g/l Na,
K tartrate) 4 ml reagent B (5 g/l CuSO4.5H2O)
4. Prepare reagent D: 6 ml phenol reagent 12 ml distilled water
5. You are now ready to begin. Add 5 ml reagent C to each tube (16 samples, 10 standards and 2
blanks). Mix. Wait ten minutes. Add 0.5 ml reagent D to each tube. Mix. Wait 30 minutes while
color develops.
6. Use the spectrometer to read the absorbance of each sample at 650 nm. Use the blank to
match the cuvettes and set the absorbance (100% T) to zero, and check this setting periodically
while making your readings. Read the standard curve tubes at the beginning and again at the
end, to get an idea of the variability of the readings.
7. Plot A versus concentration for the samples of the standard curve. Use this graph to determine
the mean concentration, in ug/ml, of your 8 diluted samples. Using the dilution factor, calculate
the protein concentrations in the original 4 samples. Duplicate determinations should agree
closely, as should different dilutions of the same original sample. (In your report, show your
calculations, data, and graph.) Multiply by the volume in each fraction to get the protein content
per fraction. Does the sum of the nuclear +mitochondrial +soluble equal the original
homogenate?
8. When finished, rinse all tubes thoroughly in running tap water and leave to drain in dishwasher
basket. (Before discarding your fractions, you will want to be sure the assay worked properly.
Otherwise you may need to repeat it!) Wash the spectrometer cuvettes carefully and leave to
drain at your station.
Part C: Assay of fractions for succinate dehydrogenase (SDH).
Introduction:
This enzyme catalyzes the oxidation of succinate to fumarate in the Krebs cycle. It is a good
choice as a marker enzyme for mitochondria because not only is it readily assayed, but also it is
the only Krebs cycle enzyme to remain bound to the inner mitochondrial membrane. Thus even if
motochondria lose some their soluble contents through damage during isolation, they remain able
to exhibit SDH activity.
The chemical reaction is given in Figure II.A.2. Flavine adenine dinucleotide (FAD) is a coenzyme
covalently bound to the SDH enzyme. For the enzyme to complete its catalytic cycle, the
electrons it receives from succinate are ordinarily passed on down the electron transport chain to
oxygen, and the reduced FAD becomes reoxidized, ready to encounter another succinate.
This procedure can be altered, however, by blocking the electron transport chain and providing
the assay system with artificial electron acceptors to draw the electrons from reduced FAD. If
these acceptors are dyes with characteristic absorbance in the oxidized and reduced form, the
progress of the reaction may be assayed by the change in the amount of color.
One very useful artificial electron acceptor is 2, 6- dichlorophenol indophenol (DCPIP) (see Figure
II.A.3). It absorbs strongly at 600 nm when oxidized, but becomes colorless in its reduced form.
Thus the basic procedure is to mix samples of fractions to be tested (enzyme) with an assay
mixture containing, among other things, substrate and DCPIP. The absorbance of the mixture is
measured as a function of time, and thus the rate of the enzyme- catalyzed reaction is
determined.
The assay medium contains
10 mM succinate (substrate)
0.02 mg/ml DCPIP (indicator dye)
10 mM phosphate buffer, pH 7.4 (to control pH)
2 mM KCN (to block the electron transport chain) 10 mM CaCl2 (to increase the permeability of
the mitochondria)
0.5 mg/ml albumin (an osmotic buffer to keep the mitochondria from bursting).
This medium is prewarmed to 37
o
C, the physiological temperature of the reaction. Then the
enzyme sample is added, and the fading of the blue color is measured as a function of time. It is
sometimes difficult to obtain accurate rate measurements of this enzyme for two reasons:
a. The enzyme undergoes an activation in the initial phases of the incubation
which interferes with observation of the reaction itself.
b. The most accurate readings of absorbance are obtained when DCPIP is
present in only small amounts. It is reduced so rapidly, however, that rapid-
response instrumentation is needed for the best readings. Once reduced, it may
be reoxidized (albeit slowly) by atmospheric oxygen.
We will try to avoid these problems by taking readings both as we first mix the reaction and after
3 minutes (to allow for activation); we will also use a higher concentration of DCPIP than might be
otherwise desirable, in order to prolong the reaction to the point that our manual methods give
reliable data.
Procedure:
For each assay follow this procedure:
1. Pipette 2.8 ml of assay medium into a Spectronic cuvette, stopper, and place in a 37
o
water
bath or heating block. Keep a cuvette of buffered sucrose on hand as a blank to set the zero on
the spectrophotometer.
2. At t =0 add 0.2 ml of your diluted enzyme preparation (see below), and mix well. Immediately
read and record the zero-time absorbance at 600 nm, and return the tube to the water bath. Avoid
shaking the tube from this point on; the DCPIP reoxidizes readily.
3. At t =3 minutes, again read and record the absorbance at 600 nm and return the tube to the
water bath.
4. At t =15 minutes take a final reading and record the absorbance.
You will repeat the procedure (steps 1-4) on the following samples:
buffered sucrose, (a "blank" enzyme-free control);
homogenate (dilute 1/2 in buffered sucrose);
nuclear fraction (dilute 1/2 in buffered sucrose);
mitochondrial fraction (dilute 1/5, 1/10, 1/25, in buffered sucrose);
soluble fraction (dilute 1/2 in buffered sucrose).
Carry out each of the 7 assays three times. You will thus have data for 21 assays. They can be
done in groups of 3 or even 6 at a time, but be sure to stagger the starting times so that there is
time to make the readings. It is also a good idea to mix the assays so that the three assays of a
single dilution are made at different times in the course of the period.
Data analysis
The t =0 readings are important, as they include a component of turbidity that varies from sample
to sample. That component is determined by subtracting the zero-time readings of the "blank"
assays from that of the sample assays. The difference is the turbidity for that sample.
This value should now be subtracted from the t =3 and t =15 readings, yielding the component
of the absorbance reading due to DCPIP.
The rate of the reaction, v, is defined as the rate of disappearance of the colored substrate, and is
proportional to the concentration of that substrate, s:

v = -ds/dt =Ks
Rearranging,

-ds/s =Kdt.
Integrating from 0 to t,


ln (so/st) =Kt

This is the equation of a straight line whose slope, K, (in units of time
-1
) represents the activity of
the enzyme.
Thus the next step is to divide the reading at t =3, (proportional to so) by that at t =15
(proportional to st) and then to take the natural logarithm of the quotient. This value is divided by t
=12 min to give K in units of min
-1
. We define a value of 0.1 min
-1
to be 1 unit of enzyme activity.
Calculate the number of units present in each sample. Correct this value for dilution and sampling
(see sample calculation in Figure II.A.4) to give the units of activity per ml of fraction. Now divide
the activity per ml of fraction by the mg protein per ml of fraction determined last time in the Lowry
assay. This value is the specific activity of the enzyme in the fraction: units of enzyme per mg
protein.
Suggestions for further study:
The biochemistry of this exercise can be further developed by using the most active fraction
(usually the mitochondrial) to perform measurements at different (lower) substrate concentrations,
while the enzyme concentration is held constant. From the resulting rate measurements Km and
Vmax may be calculated.
Other sources of mitochondria can also be used. I have heard of success using Cauliflower
florets or Dictyostelium amebae. My impression is that any reasonably metabolically active tissue
with which you are familiar and which is abundant will do nicely.
For the instructor:
EQUIPMENT
A centrifuge capable of generating the force needed to pellet mitochondria. Compound light
microscopes, and preferably phase contrast microscopes. Automatic pipetting guns (such as Clay
Adams) are helpful. Teflon homogenizers and drive motors (electric drills work rather well). Other
means of cell disruption (Waring blender, sonication, freeze-thawing, hand-held tissue grinder)
will probably work, too, but should be tested.) Spectrometers and cuvettes (a simple Spec 20 is
adequate).
SUPPLIES AND SOURCES
Biochemicals are readily available from scientific supply houses. Laboratory rats may be
purchased or obtained from colleagues as surplus. I am told that beef, chicken or pork liver from
a local abattoir or even a supermarket also works well. I tried a published procedure using
cauliflower, but it was not easy to disrupt.
RECIPES
1. Perfusion of the rat liver.
Prepare a 50 ml syringe with a 16 gauge needle and fill it with ice-cold buffered sucrose. Open
the rat along the ventral midline in two stages, first peeling back the skin, then opening the
abdominal wall (this prevents fur getting into the preparation. Make lateral incisions at the base of
the rib cage and just above the pelvis. Fold the flaps back to reveal the abdominal cavity. Move
the intestines out of the way and locate the vena cava and the hepatic portal vein.
This next part takes some practice, but even a clumsy preparation seems to work all right in the
exercise. Insert the needle into the vena cava and gently slide it up past the branch with the portal
vein. Now clip the portal vein with a scissors and steadily but gradually expel the contents of the
syringe. The sucrose will displace the blood from the liver by retrograde perfusion and will chill
the tissue, helping to diminish degradation. You can monitor your success by noticing the
blanching of the previously dark to grayish red of the liver. If necessary, you can refill the syringe
by delicately removing it from the needle while holding the needle in place. I usually puncture the
vein during this operation, though, so it may not be worth it. If worse comes to worst, you can
simply puncture the liver repeatedly on its surface at various points and inject the sucrose
directly.
When the perfusion is done, snip the liver out of the body cavity with a scissors and remove it to a
small beaker of ice-cold buffered sucrose. At this point it can be trimmed of adherent connective
tissue, if any, and cut into pieces suitable for the students to use.
2. Buffered sucrose
I find it helpful to have on hand stock solutions of mono- and dibasic phosphate salts at 0.2 to 0.5
M concentrations that can be diluted and mixed in proportion to generate the desired pH of this
and other buffered solutions.
Roger Sloboda of Dartmouth College has recently told me that this buffer can cause the
mitochondria to clump, causing them to sediment in the nuclear fraction. He suggests using 0.25
M sucrose buffered with 3 mM imidazole-HCl, pH 7.2. I plan to try this the next time I run the lab.
3. Lowry assay reagents:
Dissolve the indicated substances to the indicated concentrations in distilled water. To prepare
the BSA, it may be necessary to start with a more concentrated solution and then dilute it.
4. Assay medium:
Mix all the ingredients except the CaCl2. The phosphate buffer may be assembled from more
concentrated stock solutions (see 2 above). Once everything is dissolved, add the calcium,
dissolved in a little water. It usually precipitates; I filter before use to remove the calcium
phosphate, but I worry about the change in concentration that results. Also it tends to continue to
precipitate during the assay, making the turbidity measurements problematic. For the last two
years I have omitted the calcium, and the assay seems to work all right.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
The chief problem with this procedure is that it is sufficiently complex that students often forget to
make a critical volume measurement or to save a critical fraction for assay. Vigilance on the part
of the instructor is helpful.
The cell fractionation parameters are reasonably flexible, so that enrichment of the mitochondrial
fraction for mitochondria and for enzyme activity is usually seen. The best separations are gained
if a. the liver is well perfused so that red blood cells don't confuse the issue; b. care is taken when
removing the first supernatant not to include any of the pellet (even if it means leaving behind
some of the supernatant); and c. care is taken when removing the second supernatant to take as
much of it as possible (even if it means losing a little of the pellet).
Neither stained slides alone nor wet mounts alone give a complete view of the structures in the
various fractions; it is desirable to do both.
The Lowry procedure is reasonably foolproof. The major mistake students make is to use their
sample dilutions straight, forgetting to use only 0.1 ml in the assay. They must begin again with
fresh dilutions, so it is good if you can notice the problem promptly. It is obvious, because once
they add the phenol reagent, the tubes all turn almost black!
The enzyme assay also works quite well. If insufficient dilution is made for the samples, a large
part of the reaction can take place during the initial activation period. In such cases there is apt to
be a misleadingly small drop in absorbance between 3 and 15 minutes, complicated further by
the tendency of the DCPIP to reoxidize, especially at the interface between the liquid and air in
the tube. The instructor can monitor the course of students reactions and recommend additional
dilution of samples that appear too concentrated. This will also help by reducing the turbidity.
Title: II. B. Cell Motility: Muscle
Authors: J ason Wolfe, Biology Department, Wesleyan University Mary Lee S. Ledbetter, Biology
Department, College of the Holy Cross
Purpose: Movement is one of the fundamental processes of life. A major form of motility is that
mediated by actin- and myosin-based microfilaments. This form is most dramatically expressed in
muscle and its contraction.
Time required: a 3-hour laboratory period with an additional visit to analyze the results of the
electrophoresis, if that is used.
Level: Intermediate
References: Any standard Cell Biology textbook has fine illustrations of the ultrastructure of
skeletal muscle and diagrams of the constituent microfilament assemblies.
Introduction:
Motility is a fundamental life process. It is carried out by systems of filamentous proteins,
including actin, myosin and tubulin. This exercise concentrates on the former two components.
Certain cells are specialized to perform almost exclusively in contraction; these are referred to as
"muscle," and they function in locomotion of the organism and transport of fluids down the various
tubes of the digestive, circulatory, and glandular systems.
In vertebrates there are three distinct types of muscle, differing in function, structure, and
distribution.
1. Smooth muscle is composed of sheets of cells, each cell with one nucleus. Contraction is
involuntary. This tissue is found in the walls of blood vessels, the digestive tract and the
reproductive system.
2. Cardiac muscle, found only in the heart, is composed of uninucleate cells bound together at
dense plaques called intercalated disks. This tissue appears to be striated, or striped, owing to
the parallel arrangements of the bundles of contractile fibers. It, too, is under involuntary control.
3. Skeletal muscle, under voluntary control, is generally attached to the skeleton. In order to give
the maximum strong, directional contraction, individual cells have fused into multinucleated fibers.
Prominent striations are visible in the light microscope, due to the extreme regularity of
arrangement of the contractile proteins.
We will concentrate on examining the third type, muscle fibers from the rabbit psoas muscle. This
muscle, from the small of the back, has long fibers and little connective tissue, making it ideally
suited to studies of this kind.
Fresh muscle fibers are difficult to use since they deteriorate rapidly. We will use instead fibers
that have been treated with glycerol (glycerinated). This treatment makes the membranes
permeable, so all the low-molecular-weight material is extracted, but the proteins of the
contractile apparatus remain behind. The preparation can be stored in the freezer for long
periods. By adding solutions of different compositions to the glycerinated muscle fibers, it is
possible to identify the requirements for contraction. Occasionally relaxation can also be
demonstrated, but this function is controlled by a regulatory system usually lost after
glycerination.
Procedure: [Both authors use this exercise or a variant of it in their teaching. This description is
compiled from the two independent descriptions. The resulting set of procedures might therefore
exceed a standard 3-hour laboratory.]
1. Take a 2 cm segment of glycerinated muscle and soak it for 30 minutes in ice-cold standard
salt solution: 100 mM KCl, 5.0 mM Pipes buffer, 4 mM EDTA, 4 mM MgCl2, pH 7.0
Move the fibers and some of the salt solution to a small Petri dish set inside a larger Petri dish of
ice. Using two pins or dissecting needles, shred the fibers longitudinally until they are less than
0.2 mm in diameter. You may find it helpful to examine them under a dissecting microscope as
you work. The finer the filaments, the better the preparation.
2. Make a wet mount of a portion of the suspension with a glass slide and coverslip. Examine the
finest filaments at low and high power and record your observations in as much detail as you are
able. Drawings are particularly helpful. Seal the coverslip to the slide with hot paraffin and
examine with oil immersion. It may be necessary to withdraw some of the fluid with a filter paper
wick; if so, avoid jarring the coverslip. What magnification is necessary to resolve the banding
pattern?
Relate the structure you see to the model of a sarcomere. Draw the repeating units in the banding
pattern and identify them. The long dark transverse bands are A bands. The long light bands are I
bands. Across the middle of the A bands, you can distinguish a lighter zone, the H zone, and you
may be able to see a narrow, dense line across the middle of the I band, the Z line. The basic unit
of contractility is the sarcomere, extending from Z line to Z line.
Measure the average sarcomere length using the ocular micrometer; measure the widths of the
various bands also. If you have calibrated your micrometer, you can convert these measurements
into units of um.
---For the remaining parts of the exercise refer to this table. ---
SOLUTION COMPOSITIONS:


KCl Pipes
buffer
MgCl
2
CaCl
2
EDTA ATP Na pyro-
phosphate
pH

Solution 1: 85 mM 5 mM 5 mM 0.1 mM

7.0
Solution 2: 85 mM 5 mM 5 mM

5 mM

7.0
Solution 3:

5 mM 1 mM

100 mM 6.4
ATP solution 85 mM 5 mM 5 mM

10 mM*

7.0

* Concentrations as low as 1 mM are also effective.
3. Make a wet mount of another fibril and seal the coverslip to the slide on two parallel sides with
melted paraffin to form a chamber. Locate the fibril under the microscope and test to see if it is
attached to the slide, by drawing standard salt solution through the chamber with a wick of filter
paper. If it is not displaced, it is attached.
While observing the fibril, draw a small drop of solution 1 through the chamber. Now add a drop
of ATP. What happens?
Measure the band lengths and sarcomere lengths and prepare a drawing as before. Which have
changed? Relate the structure to the model of a sarcomere.
See if any change results by now drawing solution 2 over the fibril. From the solution
compositions what do you expect from this procedure?
4. With a fresh wet mount of fibrils make another chamber. This time add the solutions in the
order: standard salt solution, solution 2, ATP, solution 1, more ATP. What can you conclude
about the requirements for contraction of skeletal muscle?
5. It is known that one of the contractile proteins, myosin, is soluble in 0.1 M sodium
pyrophosphate at pH 6.4. Prepare a fresh wet mount of fibrils. Draw a drop of solution 3 into the
chamber. Do you observe any change in the banding pattern? Which band contains myosin? Can
extracted fibers contract? Test your prediction.
Suggestions for further study:
1. Myosin
Divide any teased muscle fiber you have finished using between two small test tubes. Label one
"total protein" and add to it a few drops of solubilizing solution (1N NaOH +0.3% sarkosyl, a
detergent). Label the other "extracted myosin" and add a few drops of solution 3. Bring them to
the instructor. He/she will combine the fluid components from the two preparations of each group,
and will then separate the proteins by electrophoresis on 5% polyacrylamide gels. The gels will
be stained for protein. Standard mixtures of known proteins will also be run on each gel.
Observe the stained gels. Each type of protein forms a band at a position determined by its
molecular weight. The distance the protein has traveled from the origin, the point at which it was
applied to the gel, is called its relative mobility, Rf. It is the ratio between the distance of the band
from the origin and the total distance a marker dye traveled during the electrophoresis. Measure
the Rf for any distinct bands you find on the gel.
There is a correlation between the log (molecular weight) and the Rf of a protein. Make a graph
expressing that correlation for the protein standards. Estimate the molecular weights of the
components in the two preparations of muscle proteins. Can you identify those components?
2. Demonstrations
Examine prepared slides of smooth, striated, and cardiac muscle. Locate the muscularis layer in
a cross section of intestine. Compare the structures of the three with each other and with your
glycerinated fresh muscle fibers.
Examine electron micrographs illustrating the fine structure of striated muscle. Also examine
fluorescent micrographs of fluorescent-antibody localization of actin and myosin in non-muscle
cells. What do these preparations reveal that standard light microscope examination does not?
View the film loops on "Muscle Contraction" and "Cytoplasmic Motility."
3. Actomyosin
You will be provided with a solution of commercial actomyosin, the monomeric form of the muscle
proteins, at a concentration of 1 mg/ml in 0.6 M KCl. Place it in a 1-ml tuberculin syringe with a
needle.
Take a clean Petri dish and fill it with a filtered solution of 50 mM KCl -5 mM MgCl2. Check it
under a dissecting microscope to be sure no threadlike particles are present. Now slowly squeeze
a part of the syringe contents through the needle into the solution. Observe carefully to see
whether threadlike precipitates form. If so, continue. If not, cover the dish and set it aside.
Examine it periodically for any change.
When threadlike precipitation becomes apparent, add a few drops of 0.1 M ATP along the thread
and record your observations. What do you conclude causes actomyosin to form a thread? What
effect does ATP have on the thread?
If you have any actomyosin left, put it into a Spectronic cuvette and add 0.6 M KCl to make 3 ml.
Read the absorbance at 540 nm (turbidity). Now add 0.1 ml of 0.1 M ATP. Mix and read the
absorbance. Continue reading at intervals during the period. What do you conclude about the
effect of ATP on actomyosin at high ionic strength? How does this model system differ in its
behavior from the glycerinated muscle fiber? from intact muscle?
For the instructor:
EQUIPMENT
Light microscopes equipped with 100x objectives and preferably phase contrast. A colleague has
suggested that a homemade polarizing microscope can be assembled using polarizing film,
inexpensively available from Edmunds Scientific. Simply place a piece over the illuminator and a
second piece over the ocular lens, adjusting to get the maximum polarizing effect consistent with
the intensity of the microscope illuminator. The film can be reused from one year to the next.
Dissecting microscopes Standard centrifuge, preferably refrigerated Gel electrophoresis
equipment
SUPPLIES AND SOURCES
Glycerinated muscle fibers can be purchased from any biological supply house (Carolina,
Connecticut Valley, Wards, etc). Biochemicals are also readily available (Sigma).
To make the contraction chambers it is helpful to have thermostatically controlled heat cans in
which to melt the paraffin. A hot water bath is a suitable alternative but avoid an open flame,
since paraffin is highly flammable. It has been suggested that fingernail polish will work too,
though it dries slowly. In a pinch you can dispense with the sealing, but it may be harder to
prevent the specimen moving during manipulations.
Myosin is rather high molecular weight so the gel electrophoresis is best accomplished in
denaturing polyacrylamide gels of relatively low concentration. These can be prepared on site or
purchased ready-made for a not unreasonable cost. Standards can also be purchased. If they are
prestained, the progress of the electrophoresis can be readily monitored, improving its
demonstration value.
RECIPES See the procedures section for the compositions of the salt solution and the various
other solutions.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
Students observing the striated muscle may count the two halves of the A band as two separate
bands. Also measuring the dimensions can be difficult even under oil immersion. A useful
strategy is to count the number of bands between two divisions of the ocular micrometer, then
use division to calculate the dimensions of a single band.
The actomyosin precipitation does not always work convincingly. It is hard to distinguish the
precipitated threads from preexisting contaminants in the salt solution. After adding ATP,
movement and apparent contraction are usually seen but could be explained by mechanical
agitation of the surface of the liquid.
Title: II. C. Cell Motility: Cilia and their regeneration Authors: J ason Wolfe, Biology Department,
Wesleyan University Mary Lee S. Ledbetter, Biology Department, College of the Holy Cross
Purpose: To demonstrate motility of Tetrahymena mediated by their cilia and the capacity of cilia
to regenerate in cells from which they have been sheared off.
Time required: 3 hours, possibly a follow-up visit to the lab later, if regeneration does not go as
fast as anticipated
Level: Intermediate
References: Wolfe, J ., "Cell division, ciliary regeneration, and cyclic AMP in a unicellular system,"
J . Cell Physiol. 82: 39-48 (1973). Rosenbaum, J .L. and K. Carlson, "Cilia regeneration in
Tetrahymena and its inhibition by colchicine," J . Cell Biol. 40: 415-425 (1969).
Introduction:
Motility is a fundamental life process. It is carried out by systems of filamentous proteins,
including actin, myosin and tubulin.
Cilia are hairlike appendages found on the surface of cells of many animals, protozoans, and
lower plants. Indeed one subdivision of Protozoa is the phylum Ciliophora, in which the presence
of cilia is a major feature in the classification of these organisms. In animals ciliated cells are
often found lining cavities, such as the trachea of mammals or the gills of mollusks. Wherever
they are found, cilia function by beating in a rhythmic motion. This motility allows ciliated protozoa
to move through their aqueous surroundings and promotes the flow of fluid past the cells of a
ciliated epithelial tissue.
Structurally cilia are about 0.25 um in diameter (see Figure II.C.1). They are covered by a
membrane that is an extension of the plasma membrane and the interior is an extension of the
cytoplasm. A chief feature of the internal structure of a cilium is its axoneme, a bundle of
microtubules and their associated proteins that extends from the base to the tip. Like all cellular
microtubules, those in the axoneme are hollow tubes 25 nm in outer diameter formed from
molecules of the globular polypeptide tubulin.
Within the axoneme, the microtubules are arranged in a distinct pattern of nine doublets in a ring
around a central pair of single microtubules. This pattern is called the "9+2" array and is
characteristic among most forms of cilia. The microtubules of the 9+2 array extend from the cilium
proper into the main part of the cytoplasm where each joins to a basal body. The basal body is a
short cylinder of parallel microtubules with the same outer diameter and ninefold symmetry as the
axoneme itself. Unlike the axoneme, however, the basal body contains a ring of nine fused
"triplet" microtrubules with no pair of microtubules at its core.
Ciliary movement is produced by the bending of the axoneme, stimulated by calcium and ATP. In
order for the bending to occur, adjacent microtubule doublets slide with respect to each other.
This sliding activity causes the cilium to move; coordinated movement produces a wave-like
motion among all the cilia which propels the liquid medium up and over the cilia. This is how most
ciliates such as the Paramecium and Tetrahymena move.
The protozoan Tetrahymena has the ability to regenerate its cilia if they are gently removed.
Therefore, these ciliates have been used to study factors which can influence ciliary growth. It
has been found that regeneration depends upon continuous protein synthesis and upon
processes promoting the assembly of ciliary tubulin subunits into microtubules. Polymerization of
microtubules depends upon a continuous exchange of tubulin monomers between the growing
microtubules and the cytoplasmic pool. The tubulin is synthesized and these precursors then
assemble themselves into microtubules.
Various drugs are known to alter the self-assembly process. Some act by inhibiting or enhancing
polymerization while others block protein synthesis. Colchicine and colcemid are two drugs that
inhibit the addition of tubulin monomers to growing microtubules. Taxol has the reverse effect,
preventing the cycling of monomers by stabilizing the microtubules and thus using up free tubulin
in the cytoplasmic pool. Cycloheximide inhibits cellular protein synthesis by blocking ribosomal
translocation along the mRNA.
Procedure:
[The procedure given is a composite of that used by J . Wolfe and that used by M. L. Ledbetter.
They do not differ in any essential detail. In both procedures a balance must be struck between
treatment gentle enough to avoid harming the cells but vigorous enough to remove all the cilia.]
1. Before beginning the deciliation procedure, you will need a concentrated cell suspension, so
that when you sample the cells later on in the experiment, you will have an adequate number to
observe. Obtain 50 ml of a late exponential culture of Tetrahymena and sample 0.5 ml to a test
tube with 0.1 ml 10% glutaraldehyde to fix them. Centrifuge the rest. While the centrifuge is
spinning, use the hemocytometer to determine the number of cells in the fixed sample, calculate
the total number of cells in the centrifuge tube, and calculate the volume necessary to suspend
those cells to a concentration of 1-2 x 10
6
cells/ml. When the centrifugation is complete, pipette
off enough supernatant to leave the correct volume of medium (but at least 2.5 ml), and
resuspend the cells. Take a drop and prepare a wet mount slide. Observe with the phase contrast
microscope in order to verify
a. that there are cells in suspension,
b. that the cells are Tetrahymena and
c. that they are swimming (i.e. ciliated).
2. The deciliation procedure must be done with attention to precise timing. Before beginning
prepare a flow chart of your plan of action and be sure that your materials and solutions are
assembled.
At t=0 add 5 ml of Solution A (10 mM EDTA in 50-100 mM Na acetate, pH 6.0). Stir rapidly.
After 30 seconds (t=30) add 2.5 ml of distilled water.
One minute later (t=90) add 0.25 ml of 0.4 M CaCl2, and stir rapidly. [A variant on this procedure
is to chill the cells, the solution A, and the distilled water, only allowing the cells to warm to room
temperature with the addition of the CaCl2, which is added as 0.5 ml of 0.1 M CaCl2.]
Thirty seconds later (t=120) draw the cell suspension up into a 10 ml syringe with an 18 gauge
needle and press out, helping to shear the weakened cilia away from the cells. Shear a second
time. At 2 1/2 minutes add proteose peptone to the tube bringing the volume to 50 ml. [A variant
on this procedure is to let the calcium-treated cells sit at room temperature for two minutes before
shearing (instead of 30 sec), to shear 3-4 times, and to add 8 ml of the cell suspension to 40 ml of
proteose peptone growth medium. At this point the cells can be incubated for 10 minutes at room
temperature to facilitate their recovery, or you can proceed as below.]
3. Centrifuge at low speed (1200 rpm) for 2-3 minutes. Carefully pour off the supernatant and add
1 ml proteose peptone to the pellet. Place the tube in a rack in a 30
o
C water bath.
Load a drop onto a hemocytometer and after 1 minute to allow for settling, observe and count the
cells in the central large square. The cells are not fixed, but if the cilia have been removed they
will not be moving. Therefore the number of cells over 1 or 2 large squares can be easily counted.
After 30 minutes and at 15 minute intervals thereafter, load the hemocytometer again and count
the non-motile cells in the squares. Eventually that number should decrease as cells regain the
ability to move.
4. For each time point calculate the percentage of non-motile cells, as follows:

Nt

NM =------- x 100 =% non-motile cells at time t,

N
o


where No is the number of non-motile cells counted at time 0 and Nt is the number of cells
counted at any given time. 100% minus the percentage of non-motile cells is the percentage of
regenerated cells.
On a graph paper plot the percentage of regenerated cells (Y axis) Vs time (X axis). From your
graph, estimate the time at which 50% of the cells are regenerated (CR50). The CR50 is a
measure of the rate of regeneration. As you can see, a higher value means a slower
regeneration. Would you expect the CR50 to increase or decrease if a) the temperature were
lowered to 5
o
C, or b) if an inhibitor of microtubule polymerization were added. Explain your
answers. Suggestions for further study: (If the lab period is 3 hours or longer, this can readily be
included in the exercise.)
After deciliating and centrifuging the cells, resuspend them in 20 ml of proteose peptone. Pipette
5 ml of the suspension into each of two test tubes, labeled 1 and 2, and two centrifuge tubes,
labeled 3 and 4. Now add inhibitors to three of the tubes as follows:
Tube Cell suspension Inhibitor
1 5 ml control, 0.1 ml H2O
2 5 ml 0.1 ml caffeine (160 mM)
3 5 ml 0.1 ml cycloheximide (50 ug/ml)
4 5 ml 0.1 ml colchicine (200 mg/ml)
What is the final concentration of each inhibitor?
Incubate the deciliated cells at room temperature, sampling periodically to determine
microscopically the degree of recovery taking place. After 30 minutes, centrifuge tubes 3 and 4
and resuspend each pellet in 5 ml of growth medium to remove inhibitor. Continue to sample all
four tubes. Recovery of functional cilia is indicated by the restoration of motility. Every 10 minutes
or so for the next hour sample the cells in each suspension and determine the percent of the
population that is motile.
NOTE: There are several ways to observe the suspensions for motility. Your choice is determined
in part by convenience and in part by the concentration of cells in your suspension: to obtain valid
data you will need to examine about 100 cells.
1. Place a drop of suspension in a hemocytometer chamber. This works best on relatively
concentrated cell suspensions, such as that prepared in the main part of the exercise.
2. Make a circular well of diameter 2 mm with paraffin on a microscope slide. This size is
approximately the field of a dissecting scope at intermediate magnification. Add a drop or two of
the cell suspension to the well. Apply a coverslip to reduce evaporation. This will give about 100
cells in a field for cells diluted as in the supplementary exercise.
3. Place a drop of suspension into a concavity slide (or depression slide), a special microscope
slide with a hollowed-out area) and apply a coverslip. This is a reasonable alternative to the
previous procedure for dilute cell suspensions.
Whichever way you choose, count motile and immotile cells in the sample and calculate the
percent that are motile.
How much time is needed for regeneration of cilia under the various conditions? How does each
inhibitor influence the regeneration process? Did removal of the inhibitor affect subsequent
regeneration of cilia? Include in your report a flow diagram of your procedure, a record of your
data, and a graphical summary of the rate of regeneration. Comment on the influence of
colchicine, cycloheximide, and caffeine.
For the instructor:
EQUIPMENT
Light microscopes; standard microscopes are adequate but phase contrast microscopes will
resolve the cilia if students are interested in making high power observations of wet mounts.
Dissecting microscopes
Standard centrifuge, preferably refrigerated
10 ml syringes with 18 gauge needles
Automatic pipetters are useful. No more accuracy is required than the simple Clay-Adams gun-
type devices, but the added convenience is a great boon.
SUPPLIES AND SOURCES Tetrahymena can be purchased from any biological supply house
(Carolina, Connecticut Valley, Wards, etc). Biochemicals are also readily available (Sigma).
RECIPES Proteose peptone medium is made by dissolving proteose peptone powder in distilled
water and autoclaving to sterilize. It can be used to maintain the Tetrahymena until they are ready
to use. To ensure sufficient oxygen exchange, put the medium into Petri dishes or in Erlenmyer
flasks filled to no more than 0.1 of capacity.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
If the Tetrahymena are handled too roughly, they will fail to regenerate cilia. This is revealed as a
plateau below 100% in the plot of % motile cells vs. time. On the other hand, if they are not
sheared sufficiently vigorously, motile cells will be present from time zero.
Title: II. D. Preparation and Reactivation of Ciliary Cytoskeleton
Authors: Esther M. Goudsmit and Charles B. Lindemann, Biology Department, Oakland
University, Rochester, MI 48309, and J ason Wolfe, Biology Department, Wesleyan University,
Middletown, CT 06457.
Purpose: To prepare intact cytoskeletons from Tetrahymena, stripped of plasma membranes and
to use these preparations to study factors influencing ciliary beat. Time required: as little as one
hour but as much as several could be spent.
Level: Intermediate
References:
J . Wolfe. J . Cell Sci. 73: 69-85 (1985).
C.B. Lindemann. Cell 13: 9-18 (1978).
U.B. Goodenough. J . Cell Biol. 96: 1610-1621 (1983).
J .S. Goltz, T.K. Gardner, K.S. Kanous and C.B.
Lindemann. Biol. of Reprod. 39: 1129-1136 (1988). A videotape of the procedure can be loaned
to interested parties.
Introduction:
See illustration provided (Figure II.D.1). [For electronic users, this illustration may be found as
Fig.
2-2 (p. 564) in R.D. Allen, "Fine structure, reconstruction and possible functions of components of
the cortex of Tetrahymena pyriformis," J . Protozool. 14: 553-565 (1967).]
Procedure:
A. Preparation of Cytoskeleton
You have been provided with early stationary phase Tetrahymena cells that have been grown on
nutrient agar plates, transferred to 10 mM Tris-HCl pH 7.4, and maintained in this medium for 20-
72 hrs at 25-28
o
C. This was done in preparation for class. You will need to prepare cytoskeletons
fresh for each of the subsequent treatments.
1) By microscopic observation confirm that cells maintained in 10 mM Tris are
alive and motile.
2) Pour approximately 10 ml of the cell suspension into a 15 ml clear plastic
centrifuge tube and chill in a refrigerator or on ice for 10-20 min. until the cells
have settled into a visible pellet. (Cold animals don't swim, so they sink.)
While you are waiting, prepare the cytoskeleton isolation-reactivation mixture,
which must be made fresh just before use. You will need 3.15 ml isolation-
reactivation mixture for each cytoskeleton preparation. Each should be made as
follows:
Into a 15 mm clear culture dish, pipette 3.0 ml of "GRM" mixture. To this solution,
add:
0.03 ml of 100 mM DTT Final conc. 1.0 mM
0.06 ml of 100 mM MgSO4 Final conc. 2.0 mM
0.03 ml of 100 mM EGTA Final conc. 1.0 mM
0.03 ml of 10.0% Triton X-100 Final conc. 0.10%
Swirl dish gently after each addition so that the solution will be well mixed. Keep
the mixture chilled until use.
3) When cell pellet is visible, very carefully remove most of the medium with an
aspirator or 10 ml pipette; remove the rest with a Pasteur pipette.
4) Add a 1.0 ml aliquot of the cytoskeleton isolation-reactivation mixture to the
cell pellet and gently suspend the cells. Then gently pour the suspension into the
culture dish containing the rest of the mixture and swirl to disperse the cells.
5) Examine a sample of the preparation with either a hemocytometer and bright-
field microscopy or a microscope slide with a ring of paraffin and a phase
contrast microscope. In either case lower the coverslip carefully. Note the
condition of the cytoskeletons. Are any motile? The detergent Triton X-100 in the
mixture dissolves cell membranes and reveals many of the underlying details of
structure. Note the arrangement of the ciliary rows, the oral cilia, the presence of
the macronucleus.
B. Reactivation of Ciliary Beat
The following procedures should be carried out within 1-2 minutes after step A4 above.
1) To the cytoskeleton preparation in the culture dish, add 0.03 ml of 0.1 M ATP
stock solution, to give a final ATP concentration of 1.0 mM. Swirl the dish gently.
Observe the preparation in the dish and remove a sample to examine in the
hemocytometer or microscope slide. Are any cilia beating? If so, how rapidly?
Are any preparations motile? The reactivation mixture contains Mg++; what
would you predict to happen to the ciliary beat if Mg++were absent?
2) Add an additional 0.03 ml of the ATP stock solution to the preparation. What is
the final concentration of ATP now? Observe the cilia. Does the rate of beating
change?
3) Add 0.01 ml of 1 mM cyclic adenosine monophosphate (cAMP).Does the rate
change? Try this also with a fresh cytoskeleton preparation. Suggestions for
further study:
4) Add 0.03 ml of 0.1 M ADP stock solution to the cytoskeletal preparation in
which ATP is already present. Observe the cells. Are there any changes in the
rate of ciliary beat? Are there any changes in the stroke?
5) Make another cytoskeletal preparation and add to it ADP alone, first to 1.0 mM
final concentration, then 2.0 mM. Expect to see a 2 to 5 minute lag period before
an effect on ciliary beat becomes apparent.
6) Progressively lower the pH by adding 0.01 ml aliquots of 0.1M HCl to a
preparation that is already beating. When the cilia all stop, what is the pH?
For the instructor:
EQUIPMENT
Compound microscopes, preferably with phase contrast optics. Alternatively one can purchase
polarizing film from Edmund Scientific. Laid over the illuminator of a standard compound
microscope, it can become a polarizer; taped to the top of the
oculars, it can become an analyzer. At a cost of less than $.50 per scope, one can have a
homemade polarization microscope. The film can be reused.
Centrifuge with swinging bucket rotor for low speeds (600 x g; 1200 rpm). Plastic test tubes, 30-
50 ml and 10-15 ml, preferably clear, not translucent, so pelleted cells may be easily discerned.
Plastic culture dishes, 15-16 mm diameter and transparent.
Small refrigerator Ice buckets, ice, aspirator, hemocytometers, slides and coverslips, paraffin
wax, 2 and 10 ml pipettes, 10-50 ul micropipettors and tips.
Microscope slides, paraffin and coverslips or hemocytometers.
SUPPLIES AND SOURCES
To obtain effective ciliary reactivation, you must use very pure distilled, deionized water. Tap-
distilled water will not do.
Cells: Early stationary phase cells grown on agar in 16 mm culture dishes
ATP: Not all sources are equally effective; we use Sigma #5394, billed as vanadate-free.
(Vanadate inhibits ciliary motility).
RECIPES
1. Preparation of cells:
a. Culture Solid growth medium (agar-proteose peptone) provides high density of
cells useful for this experiment, nuclear isolation, or conjugation. This procedure
is modified from Dobra, K.W., E.W. McArdle and C.F. Ehret "Growth kinetics of
three species of Tetrahymena on solid agar," J . Protozool. 27: 226-230 (1980).
Combine in a 500 ml flask
--4.0 g proteose peptone
--1.0 g dextrose
--0.4 g yeast extract
--2.0 ml FeCl3 (45 mM)*
--100 ml glass distilled water. Mix well.
Dissolve
--3.0 g agar in
--30 ml glass distilled water.
Add the agar to the other mixture. Bring the volume to 200 ml. Autoclave for 20
minutes.
* You may prefer to use instead 2.0 ml Fe++EDTA complex:
--3.35 g disodium EDTA
--100 ml 1% KOH
Adjust pH to 5.0 with sulfuric acid, being careful not to overshoot. Then add --2.5
g FeSO
4
7H
2
O
Adjust pH to 5.5; dilute tenfold. Store in brown bottle in refrigerator for several
months; ignore precipitate.
While still hot, pour 4.0 ml aliquots into 60x15 mm sterile plastic Petri dishes;
(wear gloves.) Once the medium has solidified, store the dishes wrapped (to
prevent drying) and upside down (to prevent condensation) in the refrigerator.
To transfer cells to the solid medium,
Add 2.0 ml sterile, liquid, 2% proteose-peptone medium to each dish of solid
medium. Flame sterilize a bacterial loop, cool it in the liquid, then use it to
transfer a loopfull of cells from an early stationary phase liquid culture or a solid
culture to the newly prepared dish.
Transfer a loop of cells to several new plates every two days. Cell cultures can
be stored in plastic covered containers kept moist with a towel on the bottom wet
with sterile distilled water.
b. Shift-down protocol (Wolfe, 1985)
Solution A: 10 mM Tris, 1.0 mM CaCl2, pH 7.4
Solution B: 10 mM Tris, pH 7.4 (NO calcium), with penicillin (150 u/ml) and
streptomycin (0.15 mg/ml)
1. Add 1.0 ml Solution A to the cells in the culture dish, mix
gently and pour cells into a 30-50 ml plastic centrifuge tube.
Rinse culture dish with 2.0 ml of Solution A and combine the
rinse with the first solution in the centrifuge tube.
2. Fill the centrifuge tube to 20 ml with Solution A. Centrifuge 4
min at room temperature (1200 rpm max; brake on).
3. Quickly and carefully aspirate the supernatant solution as
close as possible to the pellet without disturbing it. Remove the
remainder of the supernatant solution with a Pasteur pipette. The
pellet will be about 0.2 to 0.3 ml.
4. Immediately add 1.0 ml of Solution A to the pellet and
resuspend the cells by gentle shaking or flicking your finger
against the side. Fill the tube to 20 ml and centrifuge again, this
time at 4
o
C for 4 min (1200 rpm).
5. Remove the supernatant as before. Then resuspend the pellet
in 1.0 ml Solution B. Bring the suspension to 20 ml and pour it
into a clean 250-ml flask (the flask volume should be at least 10
times the cell volume). These cultures can be maintained at 25
o

to 28
o
for up to 72 hours.
2. Reactivation mixture : 0.017 M potassium glutamate, 0.098 M sucrose, 0.014 M Tris-HCl, 1
mM DTT, 0.1% Triton X-100 (w/w), 2.0 mM MgSO4, 1.0 mM EGTA, final pH 7.8.
Prepare 0.6 M potassium glutamate: use L-glutamic acid and titrate to pH 7.0 with KOH. Prepare
0.3 M sucrose.
Mix 3.0 ml of 0.6 M potassium glutamate, 35.0 ml of 0.3 M sucrose, and 0.18 g of Trizma-base.
Stir to dissolve and then adjust pH to 7.8 with HCl. Adjust final volume to 100 ml with distilled
water. Store in the cold. This solution is referred to as "GRM" mixture.
Prepare 0.1 M dithiothreitol in water. Store frozen as 1.0 ml aliquots.
Prepare 0.1 M MgSO4.* Prepare 100 mM EGTA. Neutralize to pH 7.0 using KOH. Prepare 10%
(w/w) Triton X-100.
Mix these ingredients just before use (see above); allow 3.15 ml for each cytoskeletal
preparation. * If you wish, you may omit this ingredient from the GRM in order to test the effects
of Mg++alone and in combination with ATP on the reactivation process. [See step B.1]
3. ATP 0.1 M. Titrate to neutral pH and store frozen as 1.0 ml aliquots.
4. ADP 0.1 M. Titrate to neutral pH and store frozen as 1.0 ml aliquots.
5. cyclic AMP 1 mM. Titrate to neutral pH and store frozen.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
1. Cytoskeletons stick to glass, so motility is best observed in the generous fluid volume provided
by a hemocytometer chamber. For high magnification observation of cilia, use a thin slide with
coverslip supported by a ring of paraffin. New slides from a box should be rinsed in distilled water
before use to remove impurities that lyse cells.
2. ATP must be free of vanadate, a common contaminant. Sigma #5394 is our customary choice.
Even it will not work if repeatedly frozen and thawed. It can be made up in a single batch, but
separate aliquots for each class section should be frozen.
3. If reactivations are very poor, try using less ATP (0.01 ml) or use 0.01 ml cyclic AMP as a
substitute method. Lower pH (about 7.4) might also produce better results. Impurities in the
reagents (especially the ATP) will sometimes prevent vigorous reactivations at physiological
concentrations, but will still permit detectable activity at a dilution of as much as tenfold.
ADDITIONAL THINGS TO DO WITH CYTOSKELETAL PREPARATIONS
1. Morphology
2. Cilia reactivation
3. Measurement of protein content of cells and of cytoskeletons (with the BioRad assay). To get
protein content/cell, do this in connection with cell counts.
4. SDS-PAGE of total protein and of cytoskeletal/macronuclear protein. This can be compared
later with SDS-PAGE of isolated macronuclei.
Title: II.E. CYTOSKELETAL TRANSFORMATION OF SEA URCHIN COELOMOCYTES
Authors: Paul G. Greenwood, Department of Biology, Colby College and J ohn H. Henson,
Department of Biology, Dickinson College Purpose: To illustrate aspects of microtubule and
microfilament involvement in determination of cell structure and its modification by altered ionic
conditions
Time required: 3 hours
Level: Intermediate References:
Edds, K.T. 1977. Dynamic aspects of filpodial formation by reorganization of microfilaments. J .
Cell Biol. 73: 479-491.
Henson, J .H. & G. Schatten. 1983. Calcium regulation of the actin-mediated cytoskeletal
transformation of sea urchin coelomocytes. Cell Motil. 3: 525-534.
Hyatt, H.A., M.S. Shure, & D.A. Begg. 1984. Induction of shape transformation in sea urchin
coelomocytes by the calcium ionophore A23187. Cell Motil. 4: 57-71. Introduction:
The body cavity of a sea urchin is filled with a fluid known as perivisceral fluid, which contains
(among other things) cells called coelomocytes. One of the major functions of these cells seems
to be to form clumps of cells that act as clots in wound-healing events. In the formation of these
cellular clumps, the shape of the coelomocyte is altered from its normal petaloid (rounded)
appearance to a filopoid (spiky) appearance. The filopoid shape may be necessary for proper
cellular clumping in these wound-healing responses.
It is known that changes in the shape of coelomocytes might be the result of the polymerization of
one or more cytoskeletal elements. The cytoskeleton is composed of a variety of filaments and
tubules. Two of these cytoskeletal elements include microtubules (composed of polymeric tubulin)
and microfilaments (composed of polymeric actin). Both proteins, when polymerized, have
numerous functions in a variety of both plant and animal cells. A wound generally alters ionic
conditions in the perivisceral fluid, and indeed, a change in coelomocyte cell shape can be
initiated in the laboratory by the introduction of a hypotonic solution. It is also thought that Ca2+
might play a central role in the initiation of changes in the shape of coelomocytes. Thus it is likely
that altered ionic conditions trigger alterations in the cytoskeleton.
We will investigate the role of the microtubules or microfilaments in the changes in shape of
coelomocytes, and we will investigate whether extracellular calcium ions are involved in the
initiation of the shape change. Coelomocytes will be isolated from sea urchins and induced to
change shape in a hypotonic solution. Isolated coelomocytes also will be treated with an actin
polymerization inhibitor (cytochalasin B) and a tubulin polymerization inhibitor (colchicine or
colcemid) to determine their effects on the shape change of coelomocytes. We also will
determine the role of Ca2+by varying the concentration of external Ca2+and by allowing the
ions to penetrate the coelomocyte plasma membrane with the Ca2+ionophore A23187.
Based on the information presented above, on your knowledge of the role of calcium as a second
messenger, and on your knowledge of the cytoskeleton, formulate hypotheses of how
coelomocytes might change shape. Read the instructions to the experiments below and make
predictions on the outcomes of the following experiments that would agree with your hypotheses.
Be sure to consider possible alternatives. Procedure:
I. Isolation and Observation of Coelomocytes:
1. Cut the Aristotle's lantern from the oral surface of each sea urchin, and pour
the perivisceral fluid from the body cavity into a beaker. Use one sea urchin per
student. You can expect 10 to 40 ml of perivisceral fluid per urchin
(Strongylocentrotus drobachiensis)
2. Dilute the perivisceral fluid 2:1 with anti- coagulant medium (2 parts anti-
coagulant medium : 1 part perivisceral fluid).
3. Place 5.0 ml of 0.8 M sucrose solution in a 16 x 125 mm test tube, and then
carefully fill the rest of the test tube with diluted perivisceral fluid. Continue
layering sucrose and perivisceral fluid into additional test tubes until each student
has at least one test tube.
4. Centrifuge all test tubes in the clinical centrifuge at top speed (1000-1200 x g)
for 8 minutes. The sucrose provides a cushion to protect the cells from hitting the
bottom of the centrifuge tube.
5. With a Pasteur pipette, carefully remove the coelomocytes from the interface
between the sucrose and the perivisceral fluid in each test tube, and transfer
them to a clean test tube along with about 1 ml of fluid.
6. Suspend the coelomocytes from each test tube in 8 mls coelomocyte medium
(CCM), and centrifuge all the tubes for 5 minutes at a low setting (75-100 x g) in
the clinical centrifuge. This serves to wash away residual perivisceral fluid.
7. Carefully pipette out the supernatant and discard. Resuspend the
coelomocytes in 7 mls CCM. This is your stock coelomocyte suspension.
8. Make a wet-mount of a small sample of coelomocytes, and observe the cells
with a compound or phase-contrast microscope. Are most of the cells in the
petaloid form or the filopoid form? Make illustrations of the different cell types.
Choose at least 50 cells randomly, and classify them in terms of their shape.
II. Induction of Shape Change via Hypotonic Shock:
Place a 0.5 ml sample of stock coelomocyte suspension into a microcentrifuge tube and add 0.4
ml of hypotonic shock solution. Mix the contents by inversion. Make wet-mounts of samples from
the tube, and observe and record any changes in the frequency of the different types of
coelomocytes over the next hour.
III. Effect of Cytochalasin B on Coelomocyte Cell Shape Changes: Place a 0.5 ml sample of stock
coelomocyte suspension into a microcentrifuge tube, and add 5 ul of cytochalasin B for a final
concentration of 1 ug/ml. Next, add 0.4 ml of hypotonic shock solution, mix by inversion, make
wet-mounts, and observe and record any cell shape changes in the coelomocytes over the next
hour.
IV. Effect of Colchicine on Coelomocyte Cell Shape Changes:
Place a 0.5 ml sample of stock coelomocyte suspension into a microcentrifuge tube, and add 5 ul
of colchicine for a final concentration of 20 ug/ml (or add 5 ul colcemid for a final concentration of
10 uM). Then add 0.4 ml of hypotonic shock solution, make wet-mounts, and observe and record
any cell shape changes in the coelomocytes over the next hour.
V. Effect of Ca2+and Ca2+ionophore on Shape Changes of Coelomocytes:
1. Place a 1.0 ml sample of stock coelomocyte suspension into a microcentrifuge
tube. Add 4 ul of 1.0 M CaCl2 to bring the final concentration of CaCl2 to 4 mM.
Add 3 ul of 10 mM A23187 to bring the final concentration of ionophore to 30 uM.
2. Place a 1.0 ml sample of stock coelomocyte suspension into another
microcentrifuge tube. Add 4.0 ul of 0.1 M CaCl2 to bring the final concentration of
CaCl2 to 0.4 mM. Add 3 ul of 10 mM A23187 as before.
3. Place a 1.0 ml sample of stock coelomocyte suspension into a third
microcentrifuge tube. Add 3 ul of 10 mM A23187.
4. Place a 1.0 ml sample of stock coelomocyte suspension into a fourth
microcentrifuge tube. Add 4 ul of 1.0 M CaCl2 to bring the final concentration of
CaCl2 to 4 mM.
5. Periodically remove samples of each solution, and observe and record any
shape changes that occur in the coelomocyte cells in a 60-minute period.
Presentation of Data Present
the results of your experiments as a formal scientific paper. Be sure to indicate the role (if any) of
calcium ions, actin polymerization, tubulin polymerization, and hypotonicity on coelomocyte cell
shape changes.
Suggestions for further study:
Several additional exercises are possible with coelomocytes as extensions of the above
experiments. Some of the techniques are more advanced and require some additional materials
such as fluorescence microscopes and electrophoretic apparatus, but all are relatively
straightforward in application. Two such exercises carried out in our laboratories are presented
below.
Preparation of Coelomocytes for SDS-PAGE:
Samples are made for electrophoresis from whole coelomocytes or from Triton-insoluble (0.5%
Triton X-100 in CCM) cytoskeletons prepared at the same time the above experiments are
undertaken. If the student have already had experience with SDS- PAGE electrophoresis, the
samples are loaded onto prepoured minigels and run during the laboratory. If the students have
not yet had electrophoresis experience, the coelomocyte samples are frozen and used as
"unknown" samples for a separate laboratory on electrophoresis. In either case, the gel proteins
are subsequently transferred to nitrocellulose and air- dried for immunoblotting. Protocols are
available on request.
Immunofluorescence and Immunoblotting Laboratory:
Teams of students are given antibodies to tubulin and to actin. Their job is to determine from
immunofluorescence and immunoblotting what protein is detected by which antibody, and which
protein is primarily responsible for the shape changes of the coelomocytes. Immunofluorescence
microscopy is used to examine control petaloid and filopoid cells as well as petaloid cells treated
with either colchicine or (colcemid) or cytochalasin. Antibody binding to the nitrocellulose in
immunoblots is visualized using alkaline phosphatase-conjugated second antibodies. By
combining immunofluorescence and immunoblotting into one laboratory period, excessive "dead"
time is avoided. Protocols are available on request. Whole cell negative staining of coelomocytes
is also possible for observation in the TEM at any point in the above exercises. Microfilaments
and some microtubules are readily visible in well-spread coelomocytes.
For the instructor:
EQUIPMENT Phase-contrast microscopes, if possible, or compound bright-field microscopes
Clinical centrifuge 16 x 125 mm test tubes (or other size to fit the centrifuge)
SUPPLIES AND SOURCES
We have found the coelomocyte system to be particularly good to illustrate a variety of cellular
principles and techniques. Sea urchins are easy to maintain for short periods of time in salt water
aquaria and are readily available year round from a variety of suppliers. The use of the
coelomocytes in these exercises makes acquiring fertile sea urchins unnecessary.
RECIPES
Anti-coagulant medium (0.5 M NaCl, 0.03 M EGTA*, 0.026 M KCl, 0.01 M Tris-HCl, pH 7.6)
Coelomocyte medium (0.5 M NaCl, 0.001 M EGTA, 0.01 M Tris-HCl, pH 7.5) Hypotonic shock
medium (0.001 M EGTA, 0.01 M Tris-HCl, pH 7.5)
0.8 M sucrose
Cytochalasin B (100 ug/ml)
Colchicine (2 mg/ml) or Colcemid (1 mM)
1.0 M CaCl2 in distilled water
0.1 M CaCl2 in distilled water
10 mM A23187 in dimethysulfoxide (DMSO)
* EGTA is ethyleneglycol-bis-(b-aminoethyl ether) N,N,N',N'- tetraacetic acid, a calcium ion
chelating agent.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
During the isolation, the perivisceral fluid and the anticoagulant medium should be well mixed to
avoid coagulation during the first centrifugation. If coagulation occurs, it is fastest to simply start
over with fresh perivisceral fluid. If the coelomocyte density seems low, coelomocytes isolated
from several tubes may be combined at step I.6.
Title: II. F. Cellular immunology: Lymphoid organs and the structure and distribution of their cells
Author: A. S. G. Curtis, Department of Cell Biology, University of Glasgow, Glasgow G12 8QQ,
Scotland, UK
Purpose: In this exercise you are going to examine the major lymphoid organs of the mouse and
become familiar with their anatomical positions, cellular contents and the distribution of the cells
which make up the lymphoid tissue. This is an essential pre-requisite for the next two laboratories
on cellular immunology.
Time required: 3 hours with perhaps another hour for examination of the slides which will have
been prepared. Level: Intermediate. Previous experience of vertebrate dissection and of simple
histological techniques will be helpful.
References: Any good book on human or vertebrate histology. Mishell and Shigis, Selected
methods in cellular immunology
Introduction:
Lymphocytes, which differentiate from precursor cells (derived from yolk sac, bone marrow and
foetal liver), migrate to the central lymphoid organs; these are the thymus and the gut- associated
bursa of Fabricius in birds or its equivalent (the bone marrow) in mammals. Those lymphocytes
which are processed by the thymus become T-lymphocytes and those processed by the bursa or
its equivalent become B-lymphocytes. Both T and B lymphocytes migrate to the lymphoid tissues:
blood, spleen, Peyer's patches, lymph nodes, lymphoid appendix, and tonsils. The liver, lungs
and skin are not normally thought of as lymphoid organs, but they too contain large numbers of
circulating lymphocytes, as do areas of inflammation. Lymphocytes recirculate continuously from
the blood to the lymphoid organs and return to the blood via the lymphatic vessels.
The main aims of this laboratory are
(i) to familiarize you with the anatomy, and
(ii) to examine stained smears or prints of the cell types in the various lymphoid organs.
Procedure:
1. Kill mouse by ether anesthesia or asphyxiation with CO2, followed by cervical dislocation.
Immediately (i.e. within 1 minute) obtain a drop of blood by cutting the tail. Place the drop on a
clean slide and make a blood film. Dry in air and fix for 2 minutes in methanol. Set aside to stain
later.
2. Pin the mouse out on a board and soak its skin with 50% alcohol. This prevents fur flying.
Using the illustration (Figure II.F.1) as a guide, dissect as follows:
Cut up the midline of the abdomen; keep the cut very shallow to avoid breaking into the
abdominal cavity. Pin out skin to either side.
Identify lymph nodes draining the INGUINAL (groin) region and also those draining the
AXILLARY and BRACHIAL regions. The numbers of brachial and axillary nodes will vary from
one to five or so at each location. If you are in any doubt as to identifying what is fat and what is
node, see if the "organ" will float or sink in a simple saline solution like Hanks BSS. Nodes sink;
fat floats.
3. Open the body cavity and identify SPLEEN, MESENTERIC LYMPH NODES and PEYER'S
PATCHES.
Remove spleen and mesenteric nodes to a dish containing a few ml of sterile or freshly prepared
Hank's medium.
4. Open the thorax and identify and remove the THYMUS to a second dish containing Hank's
medium. If the mice are more than five weeks old, the thymus will probably be smaller than in
younger mice and will contain fewer thymus-derived lymphocytes (thymocytes) 5. Dissect out
both femurs as a source of BONE MARROW. Clean and trim off both ends of the femur leaving
the shaft of the bone. Take up 0.5 ml Foetal Calf Serum (FCS) into a syringe and insert the
needle into one end of the bone. Expel the bone marrow cells by flushing FCS through the
marrow cavity into a third dish.
6. Prepare a suspension of the thymus cells by gently teasing them apart with a scalpel and
forceps into lml of Hank's. Be sure that the bone marrow cells also form a suspension in their
foetal calf serum.
7. Prepare films of the thymus and bone marrow cell suspension. Fix these slides as described
for the blood films. Make "prints" of nodes and of spleen by pressing the cut surface of the organ
onto a slide and allowing the adherent sample of cells to dry before methanol fixation.
Stain all fixed slides for 5-6 minutes in methyl-green pyronin. (Methyl Green stains DNA and
pyronin RNA, but neither stain is totally specific.)
Rinse in water. Dry and mount in Permount or Histoclad (Depex in European countries).
8. Examine all slides. Record the cell types present, their relative frequencies, and (in the prints)
their locations. Identify them by size (diameter) and by their staining characteristics. In the print
preparations you may get a good idea of cell distribution and may see "germinal centers."
Note differences in the various organs in the proportions of pyronin-rich cells and cells which do
not stain with pyronin. Eosinophils, recognizable by their brown granules, (see histology book)
may occasionally be seen. The lymphocytes may be small and blue with the nucleus occupying a
very large proportion of the cell, or larger with a pronounced red (pyronin stained) rim. The former
are probably quiescent, while the latter have responded to antigen with intense RNA synthesis.
Differing mouse strains and differing animal house practices may alter the proportions of reactive
cells.
The next laboratory uses different methods for assessing cell type. When you have completed it,
compare the methods you used in the two exercises and their respective reliability and utility.
9. If facilities are available, photograph your specimens through the microscope using color film.
When the prints have been developed, you can keep them in your notebook as a permanent
record. Phase contrast examination of unfixed unstained cells may also be tried, but will probably
be unrevealing.
For the instructor:
This exercise is most successful if there is an introductory discussion or instruction on how each
cell type is to be recognized. The diagnostic and forensic importance of recognition of cell types
should be emphasized.
EQUIPMENT
Dissecting boards and pins Thin gloves (if students prefer it for the dissection) A 1- or 2-ml
disposable syringe with a 25 gauge needle (for the bone marrow isolation) Slides and coverslips
Plastic petri dishes (bacteriological grade) Compound light microscope with objective
magnifications up to 95X or 100X on oil immersion For every ten students it is helpful to have a
photomicroscope, preferably one equipped with automatic exposure control.
SUPPLIES AND SOURCES
Laboratory mice can be obtained commercially. A disposal system for the carcasses is important.
Methyl green- pyronin and Hank's Balanced Salt Solution should be purchased from standard
biological suppliers. Fetal calf serum (also commercially available) is essential for the bone
marrow isolation.
Title: II.G. Cellular immunology: Use of surface markers to discriminate T and B lymphocytes
Author: A. S. G. Curtis, Department of Cell Biology, University of Glasgow Glasgow, G12 8QQ,
Scotland
Purpose: To introduce students to the concept that specific cell types bear sets of markers,
mostly surface molecules on the plasmalemma, and that these are of clinical diagnostic
importance. Some of these markers belong to the Cluster Designation sets (CD antigens),
originally established for human lymphocytes but which have very close equivalents in mice. CD
antigen typing is becoming very important in clinical immunology and in research. Specific cell
types are recognized not by the possession of a single unique marker but by possessing a unique
set of markers, even though individual markers may be shared with other cell types.
Time required: 1 day
Level: Students must have completed the previous exercise in this set and have had experience
of sterile procedures. Though the practical does not require cell culturing, there is the possibility
of appreciable bacterial growth in a warm laboratory during 1 day.
References: For cell markers see: Mishell and Shigis, Selected methods in cellular immunology
Introduction:
The importance of the lymphocyte in the induction and implementation of the immune response
has been known for many years. Not all lymphocytes are functionally alike and two major
subpopulations can be identified by their surface and functional properties. They are not,
however, morphologically distinguishable.
The exercise will involve dissecting out those organs which are important for the immune
response and which contain lymphocytes in varying proportions and at various stages of
development and in learning how cell-specific markers are recognized. The most important organ
is the BONE MARROW, containing the stem cells which give rise to all lymphocytes. The
THYMUS processes bone-marrow derived cells to give "T" lymphocytes (thymus-derived). In
birds, there is a gut-associated lymphoid organ called the Bursa of Fabricius which processes
other lymphocytes known as "B" lymphocytes. In mammals the corresponding organ is not known
with certainty. Both the thymus and Bursa of Fabricius are termed central lymphoid organs.
The remaining "peripheral" lymphoid organs contain mature T and B cells in varying proportions.
The largest of these is the SPLEEN, containing approximately equal numbers of T and B cells,
together with red blood cells, haemopoietic tissue and platelet forming cells (megakaryocytes).
The peripheral lymph nodes, blood, gut- associated Peyers' patches and appendix, and
mesenteric nodes make up the rest of the lymphoid organs. You should see most of these in this
exercise.
(1) Thymus-derived (T) lymphocytes
T-lymphocytes are the cells that are primarily responsible for cell-mediated immune reactions
such as delayed hypersensitivity and allograft rejection; they also act in most antibody producing
systems. The type of effector reactions for which they are responsible have one main
characteristic in common, viz: they can be transferred from one animal to another ONLY by living
cells and not by serum.
T-lymphocytes possess a surface antigen theta (now termed "Thy-1"), which is not found on B-
lymphocytes. Antisera against this antigen "theta"* will be used to distinguish T cells from B cells.
This is done by reacting the antiserum with mixtures of B and T cells followed by addition of
complement to the T-cell anti-Thy-1-serum complex. Complement mediated lysis can then be
detected by the trypan blue exclusion test, which allows dead cells (which take up the dye) to be
identified. This test allows one to estimate the percentage of T cells in a given population of
lymphocytes. You will use this method to estimate the percentage of T- cells in mouse thymus
and bone-marrow suspensions.
Helper T-cells have a surface antigen called CD4 in human beings and L3T4 in mice, whereas
suppressor T-cells have CD8 antigens in humans and an equivalent in mice. These and other
Cluster Designation (or CD) antigen sets can be used to estimate the percentage of helper and
suppressor cells and other special T-cell types by using the bead adherence test (Dynal beads).
(2) B-lymphocytes
B-lymphocytes are the precursors of the antibody-producing plasma cell. They have several
surface characteristics which distinguish them from T- cells, including possession of various CD
antigens in humans. Particularly important is the following:
(i) Immunoglobulin is present on the surface of many B cells, which can thus be visualized by its
reaction with anti-immunoglobulin serum conjugated to a fluorochrome. (ii) B cells have surface
receptors for the activated third component of complement (C'3). If they are allowed to react with
an antigen-antibody-complement complex, they can be visualized by the accumulation of such
complexes on their surface. However, this method will not be used in this exercise.
Procedure:
TRY TO ENSURE THAT YOU REACH END OF STAGE 1 OF SECTIONS B AND C BEFORE
THERE IS ANY PAUSE IN THE PROCEDURES
(A) Preparation of lymphocytes
(1) Kill mice by anaesthetizing in a killing jar and cervical dislocation.
(2) Pour 1 or 2 ml Ham's F10 medium plus 10% foetal calf serum (FCS) into each
of four petri dishes, labeled: THYMUS, BONE-MARROW, LYMPH NODES,
SPLEEN.
(3) Pin out the mice and dissect out the inguinal, axillary, brachial and mesenteric
lymph nodes. Note the Peyers' patches on the intestine. Place lymph nodes in
the appropriate petri dish.
(4) Remove the spleens and place in medium in its petri dish.
(5) Remove the thymus and place in medium in its petri dish.
(6) Dissect and clean the muscles from 4 femora, cut the ends of the bone and
inject 1 ml of Foetal Calf Serum (FCS) through the top end of each femur, so that
the bone marrow flows out into a petri dish. Use the syringe and 25G needle
provided.
(7) Make cell suspensions of all the other organs, passing them gently up and
down in a 10 ml or 5 ml syringe whose end (nozzle) is in the petri dish. Avoid
blowing bubbles. Use a fresh syringe for each cell type. The spleen will need
cutting into four or five bits before this process is used. Allow lumps to settle.
Transfer the cells from the petri dishes into test tubes or conical tubes and label
the tubes with your initials and the respective cell suspension's name. Keep all
suspensions stored in an ice bucket.
(8) Estimate the concentration of cells in each suspension using a
hemocytometer (except for spleen, for which see 9 below). Adjust to a final
concentration of about 10 million per ml. (See note at end about counting cells.)
Place tubes in ice when not using them. Then use cells in protocol B, C and D
below.
(9) Using the spleen suspension only, count the number of white cells using
WBC counting fluid as a diluent. To do this, fill a white blood cell pipette with
spleen suspension up to the 1 mark. Then fill to the mark above the bulb with the
purple WBC counting fluid. Shake the pipette contents and run out the pipette till
the medium in the dead space of the pipette has run to waste; add part of the
remainder to a hemocytometer. This medium lyses red blood cells and facilitates
counting. One count should also be done using trypan blue as the diluent to
estimate viability. You may need to lyse the red cells first. Multiply cell counts by
10 (the factor by which you diluted them in the pipette) to get true counts. On the
basis of this count of the white blood cell population, adjust the original spleen
suspension by adding medium to dilute (or by centrifuging to concentrate it) to a
final concentration of between about 5 million and 10 million WBC/ml. Use this
cell population in subsequent procedures as spleen cells. It will contain red blood
cells but will also have sufficient lymphocytes for useful results.
(B) Anti-Thy-1 cytotoxicity test Reagents: anti-Thy-1 antiserum and complement
(1) For each cell suspension:
(a) transfer aliquots of 0.1 ml (1 million cells) to each of two
labeled tubes. Add 0.1 ml of anti-Thy-1 antiserum to one tube
and 0.1 ml of medium to the other (control). Leave for at least
one hour in cold.
(b) Cover the tubes containing the remainder of the cell
suspension with parafilm and keep them in ice for use in
procedure D.
(2) Add 0.1 ml freshly prepared complement (1:10) to both test and control tubes.
Incubate at 37
o
C for 40 minutes.
(3) Add 0.1ml trypan blue to each tube. If the cells have settled into a pellet, very
gently resuspend them with a pipette. Wait 10 minutes.
(4) Resuspend and estimate the proportion of cells which have taken up the dye.
(Alternatively live cells can be visualized by staining with fluorescein diacetate
(1:5000 in medium) and using a fluorescence microscope with FITC filter sets.
Live cells take up the non-fluorescent diacetate and hydrolyse it to fluorescing
fluorescein.)
(5) What percentage of T-cells do you have in each of your suspensions?
Remember to subtract control values from experimental values.
(C) Detection of immunoglobulin on lymphocytes Reagent: FITC-labeled rabbit anti-mouse Fab
fragment of IgG. (Note that an anti-Ig reagent could also be possible but would recognize Ig by its
Fc part.) (Texas Red labeled reagents also work very well, but not all microscopes are equipped
with the special filter sets for this fluorochrome. Texas Red is generally brighter and fades less
than FITC)
(1) Incubate 1 ml of each cell type suspension with 0.05 ml of the reagent for
about 2 hours. To stop pinocytosis, make sure that the incubation is done at 4
o
C.
(2) Wash cells four times by repeated centrifugation and resuspension in Eagle's
medium.
(3) Finally resuspend cell pellet in 0.2 ml medium and place a drop on a
microscope slide with a No 1 coverslip on top.
(4) Examine by blue light fluorescence with a 50x objective (oil immersion) and
note ring staining, patching and capping. There will probably be an increase in
the latter two types with time. Count proportion of cells which are Ig positive. If
fluorescence is very weak, use SIT or ISIT camera under supervision.
(D) Use of antibody-bearing beads (Dynal beads) for measuring the proportion of T helper cells
(1) Add 0.25 ml of each of the lymphocyte suspensions to 0.2 ml of anti-L3T4
antibody conjugated onto Dynal beads.
(2) Incubate for 30 minutes at 37
o
C.
(3) Use the magnetic separator after instruction on its use and separate cells
bearing beads from those not bearing beads. Count the cell population density
for both fractions under a hemocytometer. Make sure that the cells bearing
beads are resuspended in an equivalent volume of medium before counting.
They will be the L3T4 positive (=CD4 positive) cells. Check that all the cells in
this fraction are beaded. They can be seen under phase-contrast microscopy as
small blue-gray spheres attached to the cells. If they are not, make appropriate
corrections to your count.
The class should compare their counts of Ig+, Thy1 - cells, which are B lymphocytes, and of Ig-,
Thy1 +, and L3T4 +cells, which are T cells, for the various organs. (L3T4 +cells are helper T
cells.) Note that there will always be a proportion of cells which do not react to any of the
reagents used here. What is the reason(s) for this? (Hint: there are at least 4 good ones.) The
class should also discuss whether they think the results obtained in this exercise are a better
method of cell type identification than the histological methods used in the earlier exercise (II.F).
Suggestions for further study:
Other fluorescent antibodies can be used to detect CD antigen equivalents or low levels of
various Igs can be used. Additional antibody markers can be tested for cytotoxicity with
complement, for immunofluorescent staining, or for their ability to promote cell separation when
derivatised to magnetic beads. Comparisons could be made between cells from younger or older
animals.
If good fluorescence results are obtained, it is nice to have the students obtain color prints for
their records. High speed color print film such as Konica 3200 ASA or Fujichrome 2000 ASA
gives good results even if the color rendition and graininess are a little poor.
For the instructor:
EQUIPMENT
Dissecting boards for mice, pins and instruments for dissection.
Refrigerator or ice buckets.
Petri dishes for holding organs and making cell suspensions.
Centrifuge (low speed) with test-tubes, suitable for centrifuging and racks.
Fluorescence microscope, preferably with automatic camera and mount for video camera.
Hemocytometers.
White blood cell counting pipettes.
Magnetic separator for magnetic beads.
Plastic or glass 20 ml capped bottles.
Pasteur pipettes with teats.
Low-light-level video camera and phase-contrast microscope (optional but useful).
SUPPLIES AND SOURCES
Mice.
Parafilm.
A saline solution such as Hanks-Hepes.
Anti-thy.1 antibody, preferably monoclonal, of an Ig class that has complement binding activity. If
a good monoclonal is available it may be used at dilutions in the range 2000 to 50,000 in saline.
Complement, diluted 1:10 or as advised in saline. Should be made up within four days of use
(preferably on the same day) from deep frozen lyophilised stock or fresh serum (non-heat
inactivated, obtained that day)
FITC- or Texas Red- (Sulphorhodamine) labeled rabbit (or other species) anti Mouse Fab IgG or
IgM.
The L3T4 labeled Dynal beads will have to be made by the laboratory, I believe, but the
technique is easy following the manufacturer's instructions. Other specific antibodies can be
derivatised to the beads instead if desired:
Dynal Beads (M-280), tosylated type.
Antibody against L3T4 antigen.
RECIPES
White blood cell counting fluid, Trypan Blue solution, and fluorescein diacetate solution recipes
are given in Mishell and Shigii.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
If the mice used are more than about 5 weeks of age, the thymus will contain almost no
lymphocytes.
If reagents are in good condition and if students obtain sufficient cells, there are few problems. If
students have 50% or less of the recommended levels of cells, then so many will be lost in
processing that they may not be able to observe any in their final test systems. Students with
poor outputs of cells should be encouraged to pool their cells with other students' cells. Antibody
activities should be tested before the practical to establish best dilutions for use. Some students
get high backgrounds in their fluorescent antibody staining reactions because they do not wash
the cells well enough. They may need instruction in draining the tube after centrifugation very
thoroughly, but without disturbance of the pellet or encouragement to wash the cells an extra
time.
Title: II. H. Cellular immunology: Mitogenic stimulation of lymphocytes
Authors: A. S. G. Curtis, Department of Cell Biology, University of Glasgow, Glasgow G12 8QQ
Scotland
Purpose: To introduce students to the mitogenic response, which is one of the early phases of
response of lymphocytes to antigen. This practical also illustrates low zone and high zone
tolerance.
Time required: 3 to 4 hours on first day. 1 hour per day on 2nd, 3rd and 4th days. Time for
automatic counting of radoactivity incorporated.
Level: This practical presumes that the preceding two practicals have been completed.
References: Chapters 2, 5, 8 and 9 from Immunology by I. Roitt, J . Brostoff and D. Male (1989).
Introduction:
T lymphocytes develop proliferative responses in response to antigens as do B cells. The
proliferative responses of B cells to a variety of lectins such as PHYTOHAEMAGGLUTININ and
CONCANAVALIN A are very rapid and strongly developed. These lectins are effective antigens.
In this practical you will use a microculture system to stimulate T lymphocytes from spleen with
Phytohaemagglutinin and you will then assess the results by detection of stimulation of S phase.
You should set up the cultures on the first day and harvest the labeled cells for counting on days
2, 3 and 4 of culture. Counting may be carried out on a scintillation counter thereafter and the
results can be made available later to the class.
NOTE THAT THE CULTURES MUST BE SET UP STERILELY so work must be carried out in the
laminar flow cabinets. NOTE ALSO that glassware as ordinarily washed and sterilized in
laboratories carries the antigen known as BACTERIAL LIPOPOLYSACCHARIDE on its surfaces,
so use of such glassware for pipetting or growing cells is likely to cause antigenic stimulation.
Fresh new plasticware must be used for all manipulations with cells in this exercise or you will
probably get non-specific stimulation.
Procedure:
Setting up cultures
1. Kill a mouse and swab fur very thoroughly with 50% alcohol. Pin the mouse on a fresh tissue
onto a cork board. Make sure your instruments are sterilized by immersion in alcohol and kept
sterile at all times. Open the mouse so that you can remove the spleen sterilely into a small
volume of fetal calf serum in Hams F10 medium.
2. Carefully and quickly cut the spleen into small pieces with a sterile pair of scissors and tease
the pieces to release the cells into the Hams F10 medium. Transfer the cell suspension without
any pieces of tissue sterilely into a plastic vial.
3. Overlay the cell suspension onto a layer of Ficoll-Hypaque (F- H) medium in a sterile 25 ml
plastic vial. (Previously you should put about 10 ml of the F-H medium into this vial so that you
can overlay with about 10 ml of the cell suspension.) Overlaying means that you very carefully
pipette the cell suspension on the top of the F-H medium so that the cell suspension, of lower
density than the F-H medium, does not mix with it. To do this, add the first few drops of cell
suspension very slowly down the side of the vial, and then slowly and carefully increase the
addition rate. If good overlaying is achieved you should see the junction of the two media as a
clear sharp, even reflective, interface (meniscus). Centrifuge the vial for 15 minutes at 2000 rpm.
4. Remove the layer of lymphocytes which will now lie at and just below the interface in the bottle
using a sterile Pasteur pipette. Leucocytes will lie further down towards the bottom of the vial and
red blood cells at the bottom of the vial. Platelets will be left in the supernatant medium.
Transfer this lymphocyte suspension to a sterile plastic vial. Add an equal or greater volume of
Ham's F10 medium plus 3% Fetal Calf serum and ITS Supplement. Centrifuge the vial for 5
minutes at 1000 rpm to sediment the cells. Resuspend the cells in a fresh lot of the Hams FCS-
ITS medium to give a cell density of about 2,000,000 per ml. This procedure washes out Ficoll-
Hypaque from the system. Check that the concentration of cells is correct using a
hemocytometer.
5. Add 0.5 ml of cell suspension and 0.5 ml of the Hams plus FCS medium to each of seven
sterile tubes.
6. Add the PHA stock to five of the tubes at the following amount: 20, 10, 4, 2, 1 ug per culture.
Keep at least two of the cultures as controls. If possible, provide several duplicates of each
concentration of PHA.
7. TAKING APPROPRIATE PRECAUTIONS, as advised by the radiation safety rules in use in
your laboratory, ADD 1.5 MICROCURIE OF TRITIATED THYMIDINE TO EACH TUBE. Reseal
them. Incubate them at 37
o
C overnight or longer as required.
Harvesting (Days 2, 3 and 4)
8. Harvest the cultures by placing a filter disc (0.45 um pore size with a prefilter) on a Millipore
filter assembly set up in the radioactive room. Add 2 ml of Hanks-Hepes medium to a tube and
vortex mix it to resuspend the cells. Decant onto the filter. Add 4 ml of Hanks- Hepes medium to
the tube and repeat the vortex mixing. Decant the wash onto the same filter. Use the vacuum line
to draw the medium through the filter. Add 10 ml of cold 10% Trichloracetic acid to the filter
assembly and draw it through. This removes TCA-soluble material. Wash the precipitate with
absolute methanol (three washes). Remove the filter and place in a numbered scintillation vial.
9. The vials will then be counted to obtain measures of the incorporation of tritiated thymidine
during S-phase so that the system provides a measure of whether entry to S-phase has been
stimulated by the PHA. You should find that low doses stimulate but that high doses tend to
inhibit thymidine incorporation.
Suggestions for further study:
Observe blast cells in cultures by presence of pyronin- staining cells of large size.
Measure cell viability in cultures.
Type reactive cells by appropriate antibodies against T cell types.
For the instructor:
EQUIPMENT
Sterile laminar flow hood(s). Low-speed centrifuge(s) for 20 ml vials. Plastic pipettes, 1ml and
10ml sizes, 20ml vials, test tubes (screw capped), petri dishes, all available from suppliers as new
sterile items. Pipetting teats or controls. 37-38
o
C incubator or hot room. Test tube racks for
upright stacking of the test-tubes used for culture. Sintered glass vacuum filtration equipment for
25mm filters from Millipore or similar supplier. Vacuum line or pump with back-flow trap.
Radioactive working area.
SUPPLIES AND SOURCES
Mice from a supplier who can supply mice reasonable free from infection.
Hams F10 medium with glutamine and antibiotic supplements, 3% fetal calf serum and Insulin-
transferrin-selenite supplement ( from Collaborative Research, Sigma or other supplier)
Phytohemagglutinin from Sigma etc in sterile solution (sterile filter for students).
10% trichloracetic acid.
0.45 micron pore size 25mm dia filters and prefilters.
RECIPES
COMMON PROBLEMS AND THEIR LIKELY CAUSE
Infection of the cultures is the most likely problem. If culture tubes are markedly acid or cloudy
when the time comes to harvest the cells, the contents should be disposed of by the instructor
following recommendations for disposing of radioactively labeled bacteria. Otherwise we have
found the exercise to be very reliable, though different students are likely to find different
concentrations of PHA giving optimal responses.
Title: II.I Cellular Immunology: Enzyme-Linked Immunosorbent Assay (ELISA)
Author: Christine A. Goodall, Department of Cell Biology, University of Glasgow, Glasgow G12
8QQ Scotland
Purpose: To acquaint students with the methods and potential of ELISA techniques
Time required: Two sessions separated by a day. Day 1: one to one-and-a-half hours; day 2: five
to six hours, including two two-hour incubation periods, during which other things may be done.
Level: Little previous practical experience required. Theoretical knowledge of autoimmune
diseases and ELISA in theory is useful. Introduction:
ELISA is a very sensitive in vitro technique for detecting antibodies and antigens. It is similar to
immunocytochemistry, which is used for the localization of tissue antigens in situ.
It is one of the most widely used assays for antibodies and is commonly used in medical labs for
diagnostic purposes. It may be used, for example, to detect the presence of anti-DNA
autoantibodies in the serum of patients with systemic lupus erythematosus (SLE), or to detect
serum levels of IgG against various infectious diseases e.g. measles, Herpes simplex virus or
toxoplasma. The ELISA assay has the advantage of being very economical in the use of reagents
and of allowing a large number of tests to be performed quantitatively in a relatively short time.
The ELISA you will perform is to detect the presence of anti- DNA autoantibodies in the serum of
NZB/NZW F1 hybrid mice. These mice have a spontaneously occurring SLE-like disease. The
basic principles of the technique are as follows (See Figure II.I.1).
1) A known quantity of antigen in saline is incubated in a plastic microwell plate. Small quantities
of the antigen become adsorbed onto the plastic surface. In today's ELISA the antigen is calf
thymus DNA, which has been denatured.
2) Free antigen is then washed away and the plate is blocked with an excess of an irrelevant
protein (e.g. BSA) to prevent any subsequent non-specific binding of proteins.
3) The antiserum (or "first antibody") to be tested is then added to the plate in a series of
dilutions. In the ELISA we will perform today the test antiserum consists of serum from NZB/NZW
F1 hybrid mice, which contains anti-DNA antibodies; sera from normal mice serve as controls.
The test antiserum binds to the antigen and unbound antiserum is washed away.
4) The "second antibody" or ligand is added. This is usually an anti-IgG which is specific for the
species from which the test antiserum was obtained. If the test antiserum is obtained from a
mouse, then the second antibody will be sheep anti-mouse IgG. The 2nd antibody is covalently
coupled to an enzyme such as horse-radish peroxidase (HRP). The second antibody binds to
antibodies in the test serum and free antibody is washed away.
5) The second antibody is then visualized by the addition of a chromogen. A chromogen is a
colorless substrate which is acted upon by the enzyme portion of the 2nd antibody to produce a
colored end-product.
6) The amount of antibody in the test antiserum is then measured by optical density scanning of
the plate in an ELISA reader.
As with all antibody staining techniques, appropriate controls must be included in the ELISA.
These should included: 1) A known positive control - a sample which definitely has the antibodies
which you are looking for in the test antiserum (+ve control). 2) A known negative control - a
sample which definitely does not have the antibodies which you are looking for in the test
antiserum. 3) A control to which you do not add any of the test antiserum, to check for non-
specific binding of the second antibody to the antigen (-ve control). 4) A row of blank wells
containing PBS or BBS on which to zero the ELISA reader.
Systemic Lupus Erythematosus (SLE)
SLE is a complex autoimmune connective tissue disease. It is more common in women than
men, affecting predominantly the 20-40 year age group, and it is related to HLA types DR3 or 4.
The disease is widespread and affects many tissues of the body. Predominantly affected areas
include the kidneys (glomerulonephritis), the joints (arthritis), and the skin (erythematous
eruptions). The characteristic histological feature of the disease is fibrinoid necrosis of the
connective tissue. In the kidney, which is the most markedly affected organ, various pathological
features are seen which appear to be due to immune complex deposition; many of the glomeruli
ultimately undergo necrosis.
An important feature of SLE is the presence of autoantibodies in the serum. Most of these
antibodies are against nuclear components, and their detection using ELISA is commonly used
for diagnosis. ELISA kits for this purpose are commercially available.
Antibodies are found to two groups of antigens: 1) Antibodies to histones and DNA, especially
dsDNA. 2) Antibodies to non-histone antigens (saline- soluble nuclear and cytoplasmic
ribonucleoproteins).
New Zealand Black New Zealand White F1 Hybrid Mouse (NZB/NZW)
The NZB/NZW F1 hybrid mouse has a spontaneously occurring autoimmune complex disease
which has various features in common with SLE and is used as an animal model for the disease.
The hybrid is formed from the NZB mouse, which develops haemolytic anemia with mild kidney
disease, and the NZW mouse, which is non-autoimmune. The hybrid animals are born clinically
normal but within 2-3 months show haemolytic anemia, have anti-red cell antibodies, anti-nuclear
antibodies and positive lupus cell (LE) tests. They have circulating immune complexes with
deposits in the glomeruli. The disease is more marked in females than in males and the animals
generally die of glomerulonephritis within a few months of developing symptoms.
The severity of the disease can be reduced by immunosuppression with cyclophsophamide,
which reduces the extent of glomerulonephritis and the amount of anti-DNA antibodies. This
suggests that the autoimmune process plays a major role in the causation of the disease.
Procedure:
ELISA protocol for the detection of anti-DNA antibodies in the serum of NZB/NZW F1 hybrids and
control mice
You have been provided with all of the reagents necessary to carry out an ELISA and 8 serum
samples from NZB/NZW F1 hybrid mice and from control mice. The samples are in tubes A- H
and have been diluted to different concentrations for you.
Using the ELISA protocol outlined below determine which samples are from the NZB/NZW mice
and which are from the control mice. If possible try to determine which of the NZB/NZW samples
have the highest concentration of antibody.
You will carry out the ELISA procedure over 2 days. On DAY 1 you will sensitize the ELISA plate
with the antigen. The antigen is denatured calf thymus DNA. On DAY 2 you will carry out the
ELISA. You have been provided with the following items and solutions:
1) 1 flat bottomed microtitre plate which has been coated overnight with 50 ug/ml poly-L- lysine.
This helps the antigen to stick to the plate. LABEL THE PLATE WITH YOUR NAME AND THE
DATE.
2) BBS/TW. Borate buffered saline, pH 8.3-8.5, with Tween 20, which is a wetting agent. This is
the washing solution which you will use to remove unbound protein from the plate.
3) 10 ug/ml calf thymus DNA, the antigen.
4) 0.5% bovine serum albumin (BSA). This is an irrelevant protein which is used to block the plate
to prevent non-specific binding of antibodies to the antigen.
5) Tubes A-H: Sera from NZB/NZW mice and control mice. They have already been diluted to the
desired concentrations for you.
6) HRP conjugated anti-mouse IgG (the second antibody). It has been diluted 1/10 in
0.2%BSA/BBS/0.05% Tween.
7) 0.2% BSA/BBS/0.05% Tween, for further diluting the second antibody.
8) Substrate buffer. This contains the chromogen.
9) 2M H2SO4. This is used to stop the further development of color after a desired point.
10) Tissues, for blotting the plate.
11) Micropipette and tips. Make sure you change the pipette tip for each serum sample and also
when emptying and washing each row of wells.
12) Elisa reader
Protocol
Day 1
1) Add 50 ul of the 10 ug/ml single-stranded calf thymus DNA to each well in the plate except for
column 1, which should remain blank. Incubate for over an hour at 37
o
C.
2) Wash 3x with BBS/TW, 200 ul per well. Shake the plate vigorously and blot face down on
tissue between each wash.
3) Add 100 ul of 0.5% BSA in BBS to each well and incubate overnight at 4
o
C.
Day 2
1) Remove your plate from the fridge and wash 3x in BBS/TW. Shake and blot between each
wash as above.
2) You are provided with 8 serum samples in tubes labeled "A"- "H". These have been diluted in
2% BSA/BBS/TW. You are also provided with a tube labeled "+ve control". Refer to the plate map
(Figure II.I.2). Leave columns 1 and 2 blank at this stage. Add 50 ul of the +ve control serum to
each well in column 3. Then add 50 ul of test serum A to each well in column 4 and the other test
sera B-H to columns 5-11 as outlined on the plate map.
3) Incubate the plate for 2 hours at room temperature.
4) Wash 5x with BBS/TW. Shake well and blot between each wash.
5) Dilute the HRP-conjugated anti-mouse IgG (the second antibody) as instructed with the
BSA/BBS/TW solution. Add 50 ul of this antibody to each well in columns 2-11. Incubate at room
temperature for 2 hours.
6) Wash 5x with BBS/TW. Shake well and blot between each wash.
7) Add 100 ul of the substrate solution to each well in columns 2-11. Incubate in the dark at room
temperature for 10-30 minutes.
8) Stop the reaction by adding 25 ul of 2M H2SO4 to each well in columns 2-11.
9) Read the optical density on the ELISA reader at 490nm.
10) Do your tests confirm that the NZB/NZW mice have anti-DNA antibodies?
Suggestions for further study:
Students might consider detecting other antigens by this general type of method and should also
estimate the possible level of antigen being detected by the method.
For the instructor:
EQUIPMENT ELISA plates. Multiwell Polystyrene plates. Automatic pipettes working in microlitre
ranges, pipette plastic tips. Elisa plate reader, preferably linked to an appropriate computer for
print out, e.g. Biorad Model 460 Microplate reader and Mac IIcx computer with printer.
(Editor's note: If this item is not available, students might be able to generate quantitative results
by serially diluting the test sera successively in the various rows of the ELISA plate and
comparing the dilution that gives a comparable intensity of color in each column.)
SUPPLIES AND SOURCES Serum from mice with SLE type disease, serum from normal mice.
Single stranded DNA from any source, e.g. calf thymus. Other reagents as below.
RECIPES Additional solutions and pre-lab methods to those listed on the lab schedule.
1) BBS (Borate buffered saline)
0.1M Boric acid
0.025M Borax (Na2B4O7.10H2O)
0.075M NaCl
H2O to 1litre; adjust pH to 8.3-8.5 This solution can be made to 4x strength but no stronger
2) Coating of plates with Poly-L-Lysine
a) Add 100 ul per well of 50 ug/ml poly-L-lysine in BBS. Incubate for longer than
1 hour at 37
o
C, then overnight at 4
o
C.
b) Wash 3x with BBS/TW, 200 ul per well.
3) Substrate (if we decide not to use the kit) a) Substrate buffer Stock solutions: 0.1M Citric acid
0.2M Na2HPO4 Buffer: Mix 11 ml 0.1M Citric acid, 14 ml 0.2M Na2HPO4 and 25 ml dist. HO. The
final pH should be 5.4-5.6 b) Dissolve 0.34 mg O-phenylenediamine (OPD) in each ml of buffer.
Add 0.4 ul H2O2 per ml just before use.
For example 4.25 mg O-PD +5 ul H2O2 in 12.5ml buffer.
COMMON PROBLEMS AND THEIR LIKELY CAUSE
None have been reported provided students check that they really have added the correct
reagents to the correct wells.