Do you the MTT assay at the same time interval after treating the cells? What is your positive control? Does it also change everytime you do the MTT assay?

Hi,

please give a more detailled rundown on your approach to help with troubleshooting.

Apoptotic cells frequently show an seemingly increased viability compared to negative control cells; this is due to increased energy demand during apoptosis (an active, highly regulated process). MTT (simplified) measures cellular respiration, which will oftenly increase during apoptosis for the reasons given.

Concerning the blank: Yes, you should account for that employing blank treatments (complete medium with MTT, but without cells), however cell-free reduction should be close to zero if everything is ok with the assay; bacterial contamination of your medium however can result in extremely high blank values.

You said you use "no cells only PBS" for blanks, do you incubate the cells in PBS, too? Cells will mandatory need an energy source (usually glucose) for MTT reduction, running the assay in PBS will give you significantly reduced product amounts.

Yes Patrick im incubating cells with MTT in PBS. Also my blank OD is around 0.5 which is very high. Please help.

Hi,

ok, first change your MTT incubation to be in cell culture medium for the reasons given. Though you should optimize MTT concentrations and incubation times for each cell line, ~ 0.5 mg/ml (final concentration) for ~ 3 h should be a good start and give you at least some degree of product.

If you get a blank OD of 0.5 when incubating only MTT in PBS, there is detinitively something going very wrong; MTT in the absence of biological metabolism or chemical reduction should be stable for the mentioned duration and OD at 595 nm (check your wavelength) should be close to zero. Do you see needle-like dye crystals in the blanks like in the wells with cells (note that this is what is expected in wells with viable cells)? I'd recommend to order new MTT and create fresh stocks solutions in freshly prepared PBS and see if the problem persists.

Be aware of pitfalls of the MTT assay resulting in a wrong estimation of viability. Stepanenko AA, Dmitrenko VV. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 2015 Dec 15;574(2):193-203. doi: 10.1016/j.gene.2015.08.009.