Terrance Grant Johns
You have to do growth curves. Set up cells at different concentrations (e.g. 500, 1000, 2000, 5000 cells per well). Then do the MTT assay every day for 7 days to determine which cell number keeps growing for the whole 7 days. Higher concentrations will stop growing earlier (e.g. day 4).
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Vilma Petrikaite
You already got a good answer. However, if your cells are growing quite fast, I would suggest to use 500, 1000, 2000 and maybe 3000 cells/well. It should be enough to grow them for 1, 2 and 3 days. Also, after adding MTT dye into cells, I would suggest to check the crystal formation after 2 or 3 hours, as sometimes 4 hour incubation duration is too long.
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