I have treated cancer cells with inhibitors of cell cycle. In addition to looking at the stage of cell cycle they are in, I want to see if there is any apoptosis induced by the treatment. But I already fixed my cells with 70% ethanol for PI staining.
Hello Aparna.
Annexin V-FITC and PI as vital dye are detecting early cell death, and both dying and dead cells, respectively. The staining is meant to be live cell state to determine the stages of cell death and viable cells escaping from the PI permeability.
Thus, disappointingly, set a new experiment, followed by live cell staining according to the protocol provided by Annexin V and PI or 7-AAD.
Best wishes.
If you are planning immunoflurescence, you can place these cells in cold methanol at -20C for 15 min, and spin down at low speed like 150g, aspirate methanol, let cells semi-dry the rest of methanol, wash once with PBS and than proceed with immuno. May be someone else tell better method
Annexin V staining is done to detect the membrane asymmetry. Phosphotidylserine is maintained on the inner leaflet by a flippase and upon cell death scramblases quickly exposure PS to the outer leaflet. Even if you fix your cells, membrane cannot be fixed and are fluid, so when the cell does not have the machinery to keep the PS on the inner leaflet they will flip to the outside as well, thus making your assay rather insensitive and unreliable. I would suggest you look for alternative methods to determine apoptosis should you absolutely have to fix yours cells, like cleaved Caspase 3 staining.
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