I am seeding 2000 OSCC cells in a 96 well plate and adding drugs at varying concentrations along with a negative control (only media). I am incubating the cells woith drugs for 96hrs and then checking the viability using MTT assay. I am getting a higher OD for the cells at lowest drug concentration than the cells in media (negative control). What should I do?
Hello Sonal,
There is a possibility that the drug could be interfering with the MTT reagent, leading to false-positive results for viability.
The drug used in the assay may be having reducing activity (like the dehydrogenase activity of the cells) that converts MTT reagent into formazan.
So, the MTT assay may not be a suitable assay for your test drug. Instead of MTT assay, you should try ATP assay. The ATP assay determines the level of cellular ATP as an indirect measure of the number of viable cells.
If you may want to confirm whether there is interference of the drug in the assay, you should observe your cells under phase-contrast imaging to check whether there is decrease in cell density before adding MTT reagent. There could be a possibility that cell death may be occurring but it may still give rise to increase in viability due to the interfering (reducing) activity of the drug with MTT reagent.
Secondly, you may want to test the drug with MTT reagent in a cell-free culture system meaning the MTT reagent could be added to the drug and then incubated for 4 hrs. If there is interference, the absorbance value would be higher than the blank (culture media only).
Best,
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