Proliferation Assay in 24 - well plate - (Oct/27/2012 )

Hi everybody,

I am a PhD student and I am mainly working on cell cultures.

Although I have some experience with cell culturing I am currently facing big problems accomplishing a proliferation assay.

To be more exact, I am trying to evaluate the effect of a drug on an adherent cell line by doing crystal violet staining and I am using a 24 well plate for seeding and treating the cells.

I seed the cells at a density of 12.500/cm2 using 1ml of 10% DMEM for each well. I change the medium in order to start the treatment 16-20 hours after seeding the cells.

However what I see then under the microscope is that the cells immediately die after changing the medium and lots of them are swimming in the supernatant having totally lost their attachment from the well ground. That is happening seconds after changing the medium and applies not only for the cells treated with the drug but also for my control cells treated with DMSO (0,01% final concentration).

The strange thing is that I haven't so far noticed something like that (or at least to that extent) when working with 96-well plates.

Could it be that I do not leave the cells attach enough in the wells? Or that I am pipetting the medium too violently causing most of the cells to detach?

Does anyone more experienced than me have any idea or has already faced a similar problem?

I would really appreciate any kind of help.

I take it that you checked the wells before adding the drug or vehicle control and saw that the cells were attached? If not - see if they are attached before starting the assay. If you leave the cells for a while after the treatment, they may attach again, which could be the solution you are after. You could also try using something like dimethylformamide to dissolve the drug.

Do you have a completely untreated control as well? - you may need this to test your pipetting strength. Ideally you should dribble the medium down the side of the well slowly to prevent you washing the cells off.

The concentration of drug and DMSO is tricky for cells and can vary quite widely dependent on the cell type and line. Some cells are very tolerant and will cope well with up to 0.5% but others will only tolerate doses lower than you are using. The effect us usually a bit more delayed though.