I am new to using MTT assay. My experiment is to treat cells and see the cell viability after 24,48,72 hrs so should I grow cells in three different plates and put MTT reagent for each one separately or there is a way to read every 24 hrs until I finish the experiment?
Hello Fatma Mohamed Fouad
You should grow cells in three different 96-well plates. Once the cells have reached 80% confluency, you remove the growth media and add media containing various concentration of the candidate compound/drug and incubate all the three plates in the incubator at 37 degree C and 5%CO2.
After 24 hours, you may perform the MTT assay on the plate marked as 24 hours. Calculate the IC50 value for 24 hours. Then after 48hours, perform the MTT assay on the plate marked as 48 hours and calculate the IC50 value for 48 hours. Similarly, after 72 hours, perform MTT assay on the plate marked as 72 hours and calculate the IC50 value for 72 hours.
So, you grow cells in three different plates and put MTT reagent for each one separately at three different time points.
Best.
Dear Fatma Mohamed Fouad
I would say that a confluence of 80% at the time of adding the tested compound is too high. Let's imagine your cells after 72 hours. Instead of studying the effect of the compound, you will study the effect of exhausted ingredients in a culture medium. The confluence should be around 40%. You should adjust the cell density to the doubling of your cells' population.
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