I am new in cell culture. Recently I performed the etoposide with different concentration on 293T cells. First I seeded 100uL of media containing 60,000 cells per well in 96 well plate and incubated overnight at 37C, 5%CO2. I changed the media in the next morning and continue incubate for another 5 hours before treated with etoposide in 0 uM - 200 uM in the media. After 24 hours incubation at 37C, 5%CO2, I carried out the Alamar Blue assay and read using flourescent spectroscopy at Ex/Em: 545 nm and 590 nm according to the manufacturer's instructions. But the readings were all zeroes for all samples ranged from 0 - 200 uM etoposide. I choose the 0 uM etoposide to auto-zero in the spectroscopy setting.
I have no idea where have gone wrong, but I suspect the cells might be all dead before I carried out the alamar blue assay. Yet, when observed under microscope the cells still there. Any opinion or advice? Thanks so much.