Hi
I intend to perform MTT assay for 24 Hrs, 48 Hrs, and 72 Hrs drug treatment. Is it ok to seed different numbers of cells for each plate (24 Hrs, 48 Hrs, and 72 Hrs)? Or is it mandatory to seed a uniform number of cells for different intervals? I believe seeding different number of cells would allow for similar confluency if not the same at different time intervals. On the contrary, seeding uniform cells would lead to different cell confluency at different intervals. I have seen the majority of people following uniform cell seeding. So I would like to ask what would be the best option and their reason for doing so.
Thank you
Hello Abhijit Mitra
Yes, it is necessary to seed uniform number of cells for different intervals of time in MTT assay. The cells for all the three plates (24h, 48h, and 72h) must be 80% confluent 24 hours after plating for drug treatment.
By seeding different number of cells, you are introducing more steps in the assay which means more variables. As far as possible keep the number of steps to a minimum to avoid errors. In MTT assay, cells treated at different time points must follow similar conditions namely, seeded with equal cell density and treated with the drug at the same time.
If you are to seed different number of cells for different time points, you would have to add the drug at each time point separately when the cells become 80% confluent which would mean preparing the drug dilutions three times that may probably lead to variations in results as compared to preparing the dilutions only once and adding to each plate at the same time.
So, seeding uniform number of cells for different intervals of time is more preferrable and accurate compared to seeding different number of cells for each time point.
Best.
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