4h is perfect time! gives the cells time to reduce MTT properly. 

From my experience, no exposure time is fixed for MTT assays and also for other in vitro tests. I usually perform a 72 hours tests with daily evaluation of the endpoints, sacrifing a plate for each time. If the toxic effect is marked after 24 hours of exposure I usually evaluate the toxicity after 2, 4 and 8/12 hours.

If you have no idea on your compounds and, in literature there are no data on similar products I suggest to move from MTT to a time-course evaluation such as the one proposed by Promega (realtime_glo-mt-cell-viability-assay) which allow you to seed cells, treat them with your products and to monitor the cell viability using a probe based on luciferase. You will decide the time endpoint without preparing and treating a lot of plates.

Hi ,

Most common incubation times are 24, 48 and 72 hours.

Every compound has its mode of action. So depends on their activity we take above intervals are common.

Hope this helps 

Hi, 

For analyzing the cytotoxic effect of any agent, you should allow your cells to complete at least one cell cycle which differs from cells to cells (ranging from 16h to 40 h). Thus you can setup your experiment accordingly. 

Thanks for all your valuable comments.

3 hours of incubation before spectrophotometric analysis. A desription of procedure of MTT test, you can read in our publications. The time of incubation of cells with MTT depends on the celltype and the tested agent

Thank you Lidia Mazur.