I would seed different amount of cells. Not necessary different for each time point, but for sure more cells for 6, 12 and 24 hours of incubation, and smaller amount for the rest ones.

Also, I am interested myself, why did you select 50, 56 and 62 hours, but did not select 48 hours?

Yes unifrom seeding and number of cells seeded is extremely important.

If your cells are highly proliferating and you want to treat for 24 or 48 hours then seed 5000 cells/well in 100uL media.

Or 2000 cells/well in 100uL mdeia for 72 hours of treatment, Note: add 200uL media for 72 h dosing time.

Maximum capacity of each well in 96 well plate is 60,000-65,000 cells.

Suppose if a cell with doubling time 24 h,

5000 cells seeded in 96 well plate

before treatment it will 10,000 cells,

After treatment,

24 h= 20,000 cells

48 hours= 40,000 cells.

If in case, 72 hours= 80,000 cells approx it will cause cell death before of over confluence and no space for growth, not authentic/accuaret result.

2000 cells per well,

4000 cells before treatment,

After treatment,

24 h= 8,000 cells

48 hours= 16,000 cells.

72 hoyrs= 32,000 cells.

I hope you got the answer.

yes I do agree that uniformity of cell seeding must be maintained proprly according to the time frame of observations.overseeding of cells will cause overconfluencing culminating in cell death.so according to me the number of cells seeded should be inversely proprtional to the time frame of observations.