cytotoxicity assays - (Jul/19/2005 )

Hi

I am testing the cytotoxicity of different compounds on cancer lines. Here's my protocol:
- trypsinize cells at 90% confluency
- seed 96-well plate w/ 5000 cells/well and incubate for 24 h
- add treatment in various dilutions and incubate for 96 h
- add MTT reagent and read absorbance after 4 h.

Now, my problem is that having repeated that study 3 times, at 3 different passages, I've obtained increasing values of absorbance which suggested that more cells grew. I also determined lowering IC50.

Did you ever observe the same?
- growth rate increases dramaticaly with passage number
- growth rate increases with seeding density (I suspect my dilutions weren't accurate enough...)
- cytotoxicity increases with growth rate

Any suggestion to standardize this experiment?

Thanks

Christ phe

I feel your seed density may be high. Did you check the doubling time for the cells? It is better to start with low seeding density. I would say 800 cells/well so that within 24 hours they will double.

The increase in cytotoxicity with the seed density may be due to the same reason.