Hello Ananya Chatterjee

You should perform a seeding density titration experiment to determine the optimal seeding density for MTT assay. In MTT assay, the cells must be in the exponential phase when you treat them to give maximum response.

Usually, in MTT assay, cells are treated when they are 70-80% confluent. At this confluence, cells still grow exponentially but nearing the end of log-phase growth. For adherent cells, they will cover most of the well, but gaps are still present.

However, Jurkat is a suspension cell line. Therefore, for suspension cells, growth progress is generally measured by recommended cell density for the cell line. Do not allow the cells to reach confluency on the final day of the read out (max 72-hour time point), which may be assessed by increasing turbidity, as the number of cells increases the culture medium appears turbid. When it reaches confluence, the cells aggregate and the growth rate slows down which may affect the results.

So, when you perform the cell titration experiment (seed 10000, 15000 and 20000 cells/well) choose that seeding density which gives you 70-80% confluence after 24 hours in a 96-well plate, as the optimal seeding density for MTT assay. However, as compared to adherent cells, suspension cells may be treated with the compound/drug 7-8 hours after plating.

Best.