We usually use MTT assay with more cervix carcinoma cell lines to detect antiproliferative effect of drugs. At the end of these measurements we get absorbance values according to produced formazan crystals what is proportional to cell viability. The measured values of treated cells are always compared to values of untreated control cells. In general, the measured absorbance values of untreated controll cells (which are comes from one cell line) are constant. In the case of C33A cancer cervix carcinoma cell line the absorbance values of controll cells are about 2700 (usually between 2500 and 3000, according to formazan producing of spreaded cells in plate, 10.000 cells/well). Nowdays I experienced that these values decreased, only about 2000 in experiments. It refers a strong decreasing of cell growth activity in the case of C33A cell line. What is the reason of this decreasing? Have somebody ever experienced similar growth activity decreasing on C33A cell line? Thank you for answers!
There may be a decrease in cell viability of control cells owing to many passages of the same cell line for a long period of time. Try performing the experiment by revival of frozen cells.
It depends by how long you perform the experiments in the plate: maybe the untreated cells grow up, reach the confluence and start to die or form clumps. I suggest to reduce the number of cells/wells if you see that the control are too confluent at the time you add the MTT.
Cheers,
Giulio
Anjana, these cells was young, the passage number were 12, 14 and 20 in the experiments. We usually passage cells maximum 30 time. The cells were take from the liquid nitrogen about 40 -45 days ago.
Dear Giulio Preta, the parameters of every experiments are constant. We spread the cells to the 96 well plate, 5000 or 10000/well (it depends on the type and size of the cells). We treat the cells on the next mornig, and we wait 48 hours incubation time. After that, we add the MTT (solved in DMSO, 5mg/ml). The time, the chemicals, the values, the cells, everything are constant for years, we do our experiments using the same methods. So I dont understand it.
There is always an explanation even if the possibilities are different. Assuming that all the parameters of the experiment are constant, It can be for example that the cells are mycoplasma contaminated so they don`t grow perfectly like before, or that this aliquot you took from liquid nitrogen had more passage that the maximum of 30 times. I suggest that before getting worried you just try another vial of cells from liquid nitrogen. Moreover what is important is the ratio between treated/untreated. Is this value also changed?
There is little change in treated/untreated ratio. The main problem that the MTT assay shows that the treated cells (only low concentration of compound) are more viable, than the untreated cells, they shows higher abs. value after the MTT assay. The aliquot was fresh before the refrigeration, it has not many passage number (maybe 5, if i remember well). I will take out new aliquot of frozen cells and I will repeat the experiments again.
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