Studying Effects Of Foetal Calf Serum On Proliferation Biology Essay

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Introduction-:

Cell proliferation can be defined as the measurement of the number of cells undergoing division in a cell culture. (Roche ) There are various ways of studying cell proliferation. It can be done by performing clonogenic assays in which cells are plated on a suitable medium and the cell proliferation is measured by the formation of the colonies after a period of time. But this can be a tedious and a lengthy procedure. Two commonly used methods for studying cell proliferation are as follows.

Crystal Violet Staining-:

One of ways to study cell proliferation is to measure synthesis of DNA which is a marker for proliferation. In this method, the cell wall is broken so that the dye can permeate inside and stain the DNA.

MTT ( 3-{4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) Method-:

Cell proliferation can also be studied by measuring the metabolic activity of the cell. This is done by adding tetrazolium salts to the cells which convert them into coloured formazan dyes. Cleavage of the dye takes place to form a coloured product by the activity of dehydrogenase enzymes which indicates that there is high level of mitochondrial activity in cells. Only viable cells are detected as only metabolically active cells are able to reduce tetrazolium salts to a coloured formazan. (M. Butler, 1996) The metabolic reduction of soluble tetrazolium salts to insoluble colored formazans has been exploited for many years. (Dominic A. Scudeiro et al. 1988)

FIGURE-: Structure of MTT and its product (Roche Applied Science)

Materials and Methods-:

Passaging of Y1 adrenocortical cells-:

The mouse Y1 adrenocortical cells which grow in a monolayer were cultured in Dulbecco's modification of Eagle's medium (DMEM) containing 10% foetal calf serum, 2mM glutamine and 100U/ml penicillin and 100ug/ml streptomycin. A mixture of trypsin (0.05%)-EDTA (0.02%) (TE) was added enough to cover the cells and the solution in the flask was swirled and then discarded. This was done to detach the cells from the substratum. Another aliquot of TE was added and the flask was kept in the gas incubator for about 5 minutes at 37Ëš C. The flask was then removed and tapped to discharge any cells that were not detached. The flask was then placed in the incubator for further 5 minutes and the tapping process was repeated. Complete medium of the same amount as that of TE was added to the cells in the flask. The contents of the flask were then decanted into a universal and then centrifuged at 1000rpm (200g) for 5min to pellet the cells. The supernatant was poured off and 10ml of CDMEM was added to re-suspend the pelleted cells by pipetting them up and down. The cells were counted using Haemocytometer. After that suspension of cells were made. In this suspension, density of about 200µl of 1.25 x 105 cells/ml cell suspension. The cells were passaged into the centre 60 wells and PBS was added in others 36 wells by using multichannel pipette. Then plates were then incubated in humidified gas incubator overnight. The cell incubations were set up in two 96-well plates and the cells were treated with medium containing 0, 1, 5, 10, and 20% v/v serum, 12 wells per treatment. The dilutions of the medium were made up in 25 ml universals. The cells in the wells were washed 3 times with PBS, using multichannel pipette and media of different serum concentrations was added. The cells were then incubated for ~72 hrs. One plate was used for crystal violet uptake and the other for the MTT assay. Various growth factors and cytokines necessary for cell growth are found in the foetal calf serum. (Naseer et al. 2009)

Table 1: Serum concentration-:

% FCS

Volume of Foetal Calf Serum (ml)

Volume of medium

(DMEM) (ml)

Total volume (ml)

0 %

0.00

25

25

1 %

0.25

24.75

25

5 %

1.25

23.75

25

10 %

2.50

22.50

25

20 %

5.00

20.00

25

Crystal Violet Staining Method-:

Cell media were removed and the cells were washed with 200µl PBS. Cells were then fixed with 200µl methanol for 15 min. the methanol was then removed and the plate was allowed to dry in air in the fume cupboard. The cells were then stained with crystal violet (0.1% solution in 200mM boric acid). After staining, the plate was washed 3 times with distilled water and the stained layer was solubilised in 50µl 10%glacial acetic acid. The plate was then incubated for 30min in the gas incubator to achieve dissolution. Finally the absorbance of the wells was read using the plate reader set to read at 540nm.

MTT Method-:

20µl MTT (5mg/ml solution in PBS) was added to each of the treated wells of the 96-well plate 4hrs before the end of the experiment. The plate was then incubated at 37°C for 4 hrs. After 4 hrs, 100µl acid-isopropanol was added to each well to dissolve blue formazan crystals I in the cell layer. The plate is then incubated at room temperature for 30min. After the formazan crystals were solubilised, absorbance of each well was measured at 570nm using the plate reader.

RESULT:

TABLE 2: Effect of foetal calf serum on cell proliferation using crystal violet

% of FCS

Absorbance at 540nm

Standard deviation

0%

0.194

0.022356

1%

0.228

0.040523

5%

0.306

0.021948

10%

0.405

0.105276

20%

0.527

0.163745

Crystal Violet Assay-:

Table 3: Effect of foetal calf serum on cell proliferation using MTT method-:

% of FCS

Absorbance at 570nm

Standard deviation

0%

0.082

0.031344

1%

0.070

0.005011

5%

0.089

0.014144

10%

0.141

0.026822

20%

0.297

0.0549

MTT Assay-:

Discussion-:

From the concentration versus absorbance graphs of MTT and Crystal violet, we observe that-:

The absorbance increased with the increase in concentration of the FCS. This shows that FCS increases the viability of the cells and that the cell proliferation is dependent on the concentration of the media. The rate of cell growth will increase till substrate limits it. The cell proliferation then becomes dependent on the other factors like availability of space to grow or amount of CO2. But we can conclude that concentration of FCS is helpful for the cell growth.

From the MTT graph, we can see that absorbance of the wells with 0% concentration of FCS was slightly higher than that of ones with 5% concentration. This might have been due to mistakenly added FCS to these wells or some extra amount of MTT added. Otherwise the absorbance goes on increasing with the increasing concentration of the FCS in the media.

From the crystal violet staining method we observe that absorbance goes on increasing with the increase in concentrations of the FCS in the media.

Finally we can conclude that the concentration of FCS plays an important role in cell proliferation and that increase in its concentration favours the growth of cells.

REFERENCES-:

Roche Applied Science, Apoptosis, Cell Death and Cell Proliferation, 3rd edition (Online)

URL: www.roche-applied-science.com/sis/apoptosis/.../manual_apoptosis.pdf

2) D.A. Scudiere, R.H. Shoemaker, K.D. Paul, A. Monks, S. Tierney, T.H. Nofziger,

M.J. Currens, D. Seniff, and M.R. Boyd; Sep,1998. Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other Tumor Cell Lines. CANCER RESEARCH 48. 4827-4833.

Butler M. (1996). Animal cell culture and technology: The basics. Oxford University Press.

Fernandez R., Vetvicka V. (2001) Methods in Cellular Immunology. Second edition.CRC Press.

Koch J., Stokstd E.L.R. (1967). The FEBS Journals (European Journal of Bio chemistry): Incorporation of 3H thymidine into nuclear and mitochondrial DNA in synchronized mammalian cells.3; 1-6.

Naseer MI., Zubair H. (2009) EFFECT OF FOETAL CALF SERUM ON CELLULAR PROLIFERATION OF MOUSE Y1 ADRENOCORTICAL CELLS IN VITRO. Pak J Med Sci 2009;25(3): 500-504.

STUDYING CELL DIFFERENTIATION OF K562 CELLS TO MEGAKARYOsCYTES/PLATELETS

Immunocytochemistry (ICC) is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. This method helps us evaluate whether or not the cells express the antigen under research.

In this experiment, PMA was employed to activate the signal transduction enzyme protein kinase (PKC). It acts as a cell growth inhibitor before inducing cell proliferation. After treatment of PMA, cells differentiated towards a megakaryocytic cell type. This was visualized by the expression of CD61 (integrin β3), a platelet cell marker.

RESULT-:

The cells that were grown in the absence of PMA did not differentiate because the they did not express CD61 marker and the antibodies could not bind to the cells for facilitating enzymatic conversion of TR substrate added to give pink color. Therefore, the cells were stained blue when haematoxyline was added. The image could not beadded as the slide was damaged before the picture could be taken.

Characteristics of cells in absence of PMA:

They are round but smaller in shape

They are closely packed

They are singular, so there is no clump present in the cells

They are shine/sparkle in presence of light

They are undifferentiated

Figure: K562 cells grown in presence of PMA

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The cells grown in presence of PMA underwent differentiation and expressed CD61 marker on their cell surface which facilitated enzymatic conversion of TR substrate detected to give pink colour. When the cells viewed in the microscope, they appeared pink in colour.

Characteristics of cells in presence of PMA:

The less number of cells are present

They are round but bigger in shape

Clumps present in cells

Different cells types- two types of cells: sphere and round shape

They are not closely packed

They are differentiated

DISCUSSION-:

The immunohistochemistry method allows the identification of cells with expression of specific surface markers in tissue. The study was carried out by indirect labelling method. The primary unlabelled anti body is added to the antigen where it binds to it. This is then followed by anti-rabbit or anti-mouse IgG according to the species of the first antibody. Because of this one remaining arm of the anti-IgG is left to bind with an IgG of the enzyme attached to, to react with the substrate. This shows that the cells with PMA were differentiated.

Immunochemical reaction for detecting CD61-:

Antibody Technology (1995)

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