Hi all, I have been doing MTT assay for a couple of months now but I have found it very difficult to get an IC50 through the readings of OD. On most occasions, I have witnessed an increase in OD as I increase my dose. My sample has reported cytotoxicity and the expected result should have been the reverse of what I'm getting now.
Is it merely troubleshooting? Or has anyone experienced this pattern as well?
Hi,
what you observed is not unusual in particular in the MTT assay. Roughly described what you measure here is cellular respiration (albeit there is some controversy on this). Your compound may cause increased metabolism rates in the cells through various stress responses; this is very prominent for inhibitors of energy metabolism (e.g. azide, uncouplers, ...).
Try to further increase your test concentrations if possible, until eventually the cells die and you observe an absorbance decrease. Also, check your cells' morphology under the microscrope to get an idea of the viability.
Also, consider that your compund may interfere with the assay chemistry (some compounds can chemically reduce MTT, resulting in apparently high viability). If everything fails, maybe try another cytotoxicity approch, neutral red retention for example is unexpensive, but similar to similar in the workflow and instrumentation required.
Regards
Hello Trinayan Barthakur
Sometimes, there could be chemical interference of the test compound. You could confirm this by measuring absorbance values from control wells without cells incubated with culture medium containing MTT and various concentrations of the test compound.
Best.
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