Etoposide Drug assay - (Mar/20/2014 )
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.
Assuming these are adherent cells, then so long as the cells were visibly spread out and not rounded into balls, then they are likely to be alive.
This implies that there is something wrong with your assay - make sure the reagent has been stored properly (away from light) and that you really did use the correct ex/em filters. I see that the peak ex is 570 nm, so if you can, try using this for excitation. Try turning up the gain on the machine, incubate for longer with the reagent, and ensure that you added the correct volumes.