i'm studying apoptosis and cell cycle arrest induced by a drug, the problem is with some concentrations, after centrifugation at 300g/10min, in the cytometer i've plus than 1000 events/second wich means that there are no cells but debris and membrane fragments. Can i centrifugate at 200g/10 min before adding PBS (cell cycle) or the binding buffer (apoptosis)?
It is really hard to tell from your description, my first thought would be that your cells are all dead when you try to collect them from the plate, therefore you only have debris. My second thought is that you are damaging them when you collect them by pipetting too hard, or using the wrong pipette. Look at the cells under a microscope before you collect them, that would give you a better idea of how they look like. If you post your protocol in detail I may be able to help you more.
Good luck.
If the difference is link to the concentration of the drug, and the other samples are fine, you induced to high cell toxicity. A can recommend to compare viability with trepan blue and to be sure to perform in the right way the staining. In this case, I can recommend Ethanol protocol and pi, more then use other detergents. We published several paper studying apoptosis induced by drugs, you can find different protocols for fresh lymphoid cell compare tumoral cell.
many thanks for your answers. The doses tested are very cytotoxic (MTT assay).
Bachir
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