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Cell Biology Practical

Cell Biology Practical

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Published by: Aveek Narain on May 03, 2011
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11/14/2012

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T he study of m ouse Y 1 aderenocortical cell proliferation using crystal violet and M T T .

1. Introduction: Cell proliferation is an key method to provide the purpose of studying important biological factors such as bioassays, toxicological tests and other carcinogenic analysis. For these analysis growth factor, serum batch testing, anti-cancer drug screening and determination of cell number is required. Reference: Animal cell culture and technology. Michael Butler.,1996, Published by Oxford University Press. Materials and Methods: Cell proliferation is a important method to study many biological activities. However for the cell proliferation, passaging is required mode to get the desired result. The two methods were used in the experiment to determine the cell proliferation is described below. Materials used for cell culture: 1) Flask. 2) Centrifuge. 3) Sterile Multi and pasture pipettes. 4) Haemocytometer. 5) Gas incubator. 6) Two 96 well plates. 7) Cell culture. Cell culture Methods: The passaging of cells in which the subculture should be removed from the monolayer culture substratum by using enzyme trypsin (0.05% or EDTA (0.02%). The passage cells should be transfer into the 60 wells in the centre of the 96 well plates by maintaining a density of 0.25% × 105 cells/well (200µl of 1.25×105 cells/ml in the cell suspension). Fill up the wells with 200µl of phosphate buffered saline (PBS). Allow them to settle for a night in gas incubator. Put the cell incubations in two 96 well plates. Next cells was treated with medium containing 0,1,5,10 and 20% v/v serum which is in 12 wells. Medium should be dilated containing serum and required 3ml of each in treatment per 96 well plates ( 200µl/well). The cell should be washed 3 times with PBS by using multichannel pipette and media containing different serum should be added. The two different plates where as one should be used for crystal violet uptake and another for MTT assay it was putted for 48 hours incubation. 1 8) Trypsin/EDTA (TE). 9) Dulbecco's modification of Eagle's medium (DMEM). 10) Phosphate Buffer saline (PBS).

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The plate was incubated for 30 minutes in gas incubator to achieve the dissolution.Converted from Word to PDF for free by Fast PDF -.www. Material Used: 96 well plates for cell culture. 5 different concentrations were prepared in the following composition and was incorporated into those 60 central wells of the plate (12 wells Per treatment) and was incubated for 48 hours.com -- After 48 hours incubation cell cultures from the 60 central well of the 96 wells plates were removed and the wells were washed with 200µl PBS. The cell cultures from the 60 central wells of the 96 well plate incubated earlier were removed and the wells were washed 3 times with PBS using Pasteur pipette. The plates were then washed three times with distilled water and the stained cell layer was solubilised in 50µl glacial acetic acid. Methanol. A graph was plotted using the resulted mean. After drying the plates were stained with 200µl crystal violet per well for 20 minutes. 2 . Pasture and multichannel Pipette. Foetal calf serum (FCS). Crystal Violet. Glacial Acetic Acid. The absorbance of the wells were later read using plate reader at 540nm. Cells were then fixed with 200µl methanol for 15 minutes. Then methanol was removed and the plates were removed and the plates were allowed to dry in the air in the fume cupboard.A) Staining with Crystal Violet: This method is based on the principle of estimating cell proliferation by calculating the absorbance after staining the culture cells with crystal violet which is taken up by the viable cells in medium with different concentration.fastpdf. The absorbance of the cells of different concentration were taken and their mean was calculated. Phosphate Buffered Saline (PBS). Medium with serum. Medium. -. Gas incubator.

D.Table: 1 Composition of different serum concentration in the media: S. It was noted that the colour of cells appeared as pink after the experiment.385 Serum Conc. After 4 hour the medium was removed by using pasture pipette.7 0.618 0.1 1 0.3 1. at 540nm 0. Then it was incubated at room temperature with the lid of the plate on for 30 minutes.3 0.5 1.047 1. B) MTT method: MTT should be added 20µl (5 mg/ml solution in PBS) to each of the treated well of the 96 plate for 4 hours. 1 2 3 4 5 Concentration of FCS 0% 1% 5% 10% 20% Mean O. Then it was kept again the 96 well plate for 4 hours into the gas incubator at 37°c.2 1.No.789 0.Converted from Word to PDF for free by Fast PDF -.4 1. Blue formazan crystal was solubilised and the absorbance of the each well was taken at 570 nm by using plate reader. vs Avg OD Crystal violet method Avg OD @ 540nm 1. The 100µl of acidisopropanol was added to each well to dissolve the formazan crystal in the cell layer.2 0.964 1.www.com -- Result: It can be noted from the graph that it is a continuous increase of cell proliferation in the presence of serum upto 20%.4 0.9 0.6 0.1 0 0 1 5 10 20 Serum Concentration -.fastpdf. 3 .8 0.5 0.

It was observed that the cells appeared in sky blue colour because of the addition of formazan crystal.311 0. at 570nm 0. economical as well as safe. Two other methods used in the determination of cell proliferation.458 Serum Concentration vs Avg OD MTT method Avg OD @ 570nm 0.126 0.3 0.240 0. The maximum growth was attained around 20%. 4 .No. Discussion: It was noticed that there is no decrease rate of cell proliferation in the experiments.5% of serum concentration.Converted from Word to PDF for free by Fast PDF -.fastpdf. 1 2 3 4 5 Concentration of FCS 0% 1% 5% 10% 20% Mean O.www.1 0 0 1 5 10 20 Serum Concentration -.2 0.com -- Results: It can be noted that rate of proliferation was increased after 1.4 0. There are various methods to determine the cell proliferation which has become a common practice in scientific laboratories.D.Table 2: Composition of different serum concentration in the media.5 0. However the method should be easy. S. 2.157 0. With this it can be interpreted that the increase in the concentration of FCS increased proliferation to an extent that can be considered as optimum concentration beyond which increase of concentration may give a negative or no effect on the cell proliferation.

www. Proliferation assays are performed by culturing cells in micrititer well plates or trays followed by neutral Red staining. The continuous monitoring of cells in culture is therefore permitted. almarBlueTM monitors the dropping environment of the proliferating cells. the cells under study can be returned to culture or used for other purposes including histological studies. which can also function as a tumour promoter. Results obtained by this method in the measurement of cells proliferation in response to the cytokines IL-2 and IL-6 compare favourably with results obtained by measuring 3H-thymidine incorporation .Converted from Word to PDF for free by Fast PDF -. or alternatively.com/content/literatureContent/PDFs/alamarbluebooklet. proliferation measurement with almarblue may be made flurometrically. Phorbol Myristate acetate (PMA).pdf -. CD45RB. On the other hand almarBlueTM does not interfere with the ability of hybridomas to secrete antibody. The absorbance of the Neutral Red stained cells monitored at 550 nm is indicative of the cell number.com -- Immunocytochemistry: 1) Cell Differiention of K562 cells to megakaryocytes: The K562 ( Chronic myelogenous leukemia cell line) has been comprehensively used in the studies of cell differentiation. CD4. Spectrophotometric microtitric well plate reader and spectroflurometric microtiter well plate reader could be used.fastpdf. Reference: http://www. 5 . The cells under study may be in suspension or adherent. As with tetrazolium salts. Because almarBlueTM is non-toxic. The differiention process was monitored by the expression of platelet cell marker and also vitronectin receptor-CD61. Specifically. and heat stable antigen are found to produce similar numbers of viable cells and antigen expressing cells as non-almarBlueTM exposed cells.a) almarBlueTM Reduction: A new method for measuring cell proliferation has in recent times become accessible which uses almarBlue TM. b) Neutral Red uptake method: The quantitation of Neutral Red also has utility in measuring cell proliferation. Cells grown in the presence of almarBlueTM and subsequently analysed by flow cytomeytry for CD44. almarBlueTM does not alter the viability of cells cultured for various times as monitored by Trypan Blue exclusion.biosource. Proliferation may therefore be monitored with almarblue using a standard spectrophotometer or by spectroflurometer. stable in culture medium and is non-toxic. was used in the experiment to differiention of K562 cells to megakaryocytes. AlmaeBlueTM is soluble. Proliferation measurements with almarBlueTM may be made of eigther spectromhotometrically by monitoring the absorption of almarBlueTM supplimented cell culture media at two wavelengths.

Turner. Incubating and washing was done in between each treatment.Johnstone and M. Cornaglia-Ferraris P. 1999. Javois. -. Lorette C. 3. From this could be noted that CDDP can induce the terminal differentiation in K562 cells. two slides were prepare by cytospin. A. The effects of PMA on intracellular signalling pathways PMA is a common practice which is used to stimulate NADPH oxidase activity in the cells. Reference: Immunochemical Methods and Protocols. Both the slides were then treated with rabbit anti mouse IgG and Alkaline phosphatase anti-alkaline phosphatase complex ( APAAP ) respectively. It was then washed with TBS and mounted in Glycerol/TBS and viewed under microscope. This can be confirmed as the cells with complex only stained pink. The slides were then treated with fast red TR substrate. Published by Humana Press Inc. These cell produces haemoglobin. 2) Agents used to induce differentiation in cell culture: • Sodium butyrate • Hemin • Butyric acid • Retinoic acid.P. Results: It was noticed that cells which were incubated with PMA and treated with CD61 produced pink colour. Immunochemistry 2.67(2):142-8.Cisplatin etc. When cells treated devoid of PMA did not show pink. It indicates the cell proliferation. . 2nd ed. Franchini E. NADPH oxidase complex is recognised as a primary source of ROS (Radio active oxygen species). 1988.W.www. 6 . Both the slides were then treated with mouse anti-human CD61. The slides were washed and then counter stained with haematoxylin. CD61 inhibits the cells. Boll Ist Sieroter Milan.RAM attaches to CD61 and APAAP attaches to RAM. Published by Oxford University Press. Reference: Cisplatin-induced erythroid differentiation in K562 cells: modulation of transferrin receptor. Tonini GP.Parodi MT.. Biologists demonstrated that it could be used for the treatment for solid cancer.fastpdf.Converted from Word to PDF for free by Fast PDF -. can down the regulation of transferrin receptor which is highly expressed on the cell surface. Bologna R. A Practical Approach. In the process of differentiation at the early stage. moreover the Tf receptor activity is related with the cell growth which could be down regulated and lose terminal differentiation.Method: All were incubated in presence and in absence of PMA. This can be recognised by the addition RAM and APAAP..com -- The cell line K562 is a human erythrolekemia which can be induced to erythroid terminal differentiation by the alkylting agent as Cisplatin. In the presence of PMA.

myeloperoxide catalyses the formation of hypochlorous acid while superoxide may also react with nitric oxide to form another relatively reactive molecule as peroxynitrite. Hydroxyl radicals are extremely reactive. pp. This reaction is catalysed by NADPH oxidase. Issue of July 2. ROS are generally useful as signalling molecules and in animal as well as plant host defence.www. No. 27. Jiang Liu. Vol. ROS especially hydrogen peroxide are the main signalling molecule.The formation of superoxide anions. Joseph I. produces hydrogen peroxide.. cascade of ROS production is likely. For the further reduction of oxygen.fastpdf. which is formed by the reduction of the one-electron of molecular oxygen. However on the other hand they might produce cellular damage if they produce in an uncontrolled manner. with a short half life and react with the first molecule. Reference: Intracellular Reactive Oxygen Species Mediate the Linkage of Na /K -ATPase to Hypertrophy and Its Marker Genes in Cardiac Myocytes. 1999 -. Peter Kometiani.Zijian Xie‡.com -- 7 . 19323–19328. Jie Li. and Amir Askari. The primary ROS is superdioxide. Thereafter further reaction may lead to the formation hydroxyl radicals. in the presence of metal ions through the Fenton reaction. PMA stimulates NADPH oxidase ROS (signalling molecule) ROS has also some direct effects as induction of gene expression and involvement of phosphorylation. In neutrophils.ROS are the species which are in a more reactive state than molecular oxygen and therefore is reduced to varying degrees.Converted from Word to PDF for free by Fast PDF -. 274. Shapiro. It also alter the redox status of the cell.

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