I always do it in a cell incubator, and it works well.

We are using CO2 incubator for the same.

Initial incubation in cell culture incubator. Remember what you are measuring, i. e., metabolic activity. In the assay, NADPH-dependent cellular oxidoreductase enzymes are reducing the tetrazolium dye MTT to its insoluble formazan. This works best under conditions favorable for cellular respiration.