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. 

Hi, Andras. First of all, I would like to ask how you get 2000-3000 values on absorbtion when it is assumed that getting more than 1 in absorbance is already an unsignificant value (or you talk about cell number which was estimated by using calibration curve?). So, the values of absorbance should be below 1 firstly. Secondly, why do you dissolve or dilute MTT in DMSO? We dissolve MTT in 1x PBS. Moreover, what is the rate o cell proliferation when unaffected? I ask in order to understand if cells do not overgrow during the period you choosed.

Going further, read my article, you will understand, that first of all, you measure cell methabolic activity of cells since MTT is based on methabolic pathways, rather than cell number. Scientists trying to get easier results started to use method for cell viability to be measured indirectly, but if you affect cells by side effect, you will decrease or increase cell methabolic activity according to the type of the side effect which will lead to misinterpretation of cell viability.

In order to answer your question i would comment this way: i never get the same values of different repetitions of the same experiments. What we do, we always take control and normalise the results to control. The results of absorbtion tend to jump higher or lower in my opinion due to different cell methabolic activity that are used for experiment. This may definitely be affected by a numerious attributes- cell size, stage of cell cycle, trypsinisation etc.

Good luck