Cell proliferation can be defined as the measuring of the figure of cells undergoing division in a cell civilization. ( Roche ) There are assorted ways of analyzing cell proliferation. It can be done by executing clonogenic checks in which cells are plated on a suited medium and the cell proliferation is measured by the formation of the settlements after a period of clip. But this can be a boring and a drawn-out process. Two normally used methods for analyzing cell proliferation are as follows.
One of ways to analyze cell proliferation is to mensurate synthesis of DNA which is a marker for proliferation. In this method, the cell wall is broken so that the dye can pervade indoors and stain the Deoxyribonucleic acid.
Cell proliferation can besides be studied by mensurating 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 topographic point to organize a colored merchandise by the activity of dehydrogenase enzymes which indicates that there is high degree of mitochondrial activity in cells. Merely feasible cells are detected as merely metabolically active cells are able to cut down tetrazolium salts to a colored formazan. ( M. Butler, 1996 ) The metabolic decrease of soluble tetrazolium salts to insoluble coloured formazans has been exploited for many old ages. ( Dominic A. Scudeiro et Al. 1988
Materials and Methods
Passaging of Y1 adrenocortical cells
The mouse Y1 adrenocortical cells which grow in a monolayer were cultured in Dulbecco ‘s alteration of Eagle ‘s medium ( DMEM ) incorporating 10 % fetal calf serum, 2mM glutamine and 100U/ml penicillin and 100ug/ml streptomycin. A mixture of trypsin ( 0.05 % ) -EDTA ( 0.02 % ) ( TE ) was added plenty to cover the cells and the solution in the flask was swirled and so discarded. This was done to detach the cells from the substrate. Another aliquot of TE was added and the flask was kept in the gas brooder for about 5 proceedingss at 37Es C.
The flask was so removed and tapped to dispatch any cells that were non detached. The flask was so placed in the brooder for farther 5 proceedingss and the tapping procedure was repeated. Complete medium of the same sum as that of TE was added to the cells in the flask. The contents of the flask were so decanted into a universal and so centrifuged at 1000rpm ( 200g ) for 5min to pellet the cells. The supernatant was poured away and 10ml of CDMEM was added to re-suspend the pelleted cells by pipetting them up and down.
The cells were counted utilizing Haemocytometer. After that suspension of cells were made. In this suspension, denseness of about 200µl of 1.25 ten 105 cells/ml cell suspension. The cells were passaged into the Centre 60 Wellss and PBS was added in others 36 Wellss by utilizing multichannel pipette. Then plates were so incubated in humidified gas brooder overnight. The cell incubations were set up in two 96-well home bases and the cells were treated with medium incorporating 0, 1, 5, 10, and 20 % v/v serum, 12 Wellss per intervention. The dilutions of the medium were made up in 25 milliliter universals.
The cells in the Wellss were washed 3 times with PBS, utilizing multichannel pipette and media of different serum concentrations was added. The cells were so incubated for ~72 hour. One home base was used for crystal violet consumption and the other for the MTT check. Assorted growing factors and cytokines necessary for cell growing are found in the fetal calf serum. ( Naseer et al. 2009 )
Crystal Violet Staining Method:
Cell media were removed and the cells were washed with 200µl PBS. Cells were so fixed with 200µl methyl alcohols for 15 min. the methyl alcohol was so removed and the home base was allowed to dry in air in the fume closet. The cells were so stained with crystal violet ( 0.1 % solution in 200mM boracic acid ) . After staining, the home base was washed 3 times with distilled H2O and the stained bed was solubilised in 50µl 10 % glacial acetic acid. The home base was so incubated for 30min in the gas brooder to accomplish disintegration. Finally the optical density of the Wellss was read utilizing the home base reader set to read at 540nm.
20µl MTT ( 5mg/ml solution in PBS ) was added to each of the treated Wellss of the 96-well home base 4hrs before the terminal of the experiment. The home base was so incubated at 37 & A ; deg ; C for 4 hour. After 4 hour, 100µl acid-isopropanol was added to each well to fade out bluish formazan crystals I in the cell bed. The home base is so incubated at room temperature for 30min. After the formazan crystals were solubilised, optical density of each well was measured at 570nm utilizing the home base reader.
From the concentration versus optical density graphs of MTT and Crystal violet, we observe that:
- The optical density increased with the addition 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 growing will increase till substrate bounds it. The cell proliferation so becomes dependant on the other factors like handiness of infinite to turn or sum of CO2. But we can reason that concentration of FCS is helpful for the cell growing.
- From the MTT graph, we can see that optical density of the Wellss with 0 % concentration of FCS was somewhat higher than that of 1s with 5 % concentration. This might hold been due to erroneously added FCS to these Wellss or some excess sum of MTT added. Otherwise the optical density goes on increasing with the increasing concentration of the FCS in the media.
- From the crystal violet staining method we observe that optical density goes on increasing with the addition in concentrations of the FCS in the media.
- Finally we can reason that the concentration of FCS plays an of import function in cell proliferation and that addition in its concentration favours the growing of cells.
- Roche Applied Science, Apoptosis, Cell Death and Cell Proliferation, 3rd edition ( Online ) Uniform resource locator: www.roche-applied-science.com/sis/apoptosis/ … /manual_apoptosis.pdf
- 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 civilization and engineering: The rudimentss. 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 chemical science ) : Incorporation of 3H thymidine into atomic and mitochondrial Deoxyribonucleic acid in synchronised mammalian cells.3 ; 1-6.
- Naseer MI. , Zubair H. ( 2009 ) Consequence OF FOETAL CALF SERUM ON CELLULAR PROLIFERATION OF MOUSE Y1 ADRENOCORTICAL CELLS IN VITRO. Pak J Med Sci 2009 ; 25 ( 3 ) : 500-504.