PI staining - (Oct/17/2006 )

Hi,

When you use PI staining method for cell cycle analysis (including subG1, G1, S/G2/M), do you also collect the medium and pellet the possible cells in the medium for analysis? My purpose is to detect how many cells in apoptosis, will apoptotic cell detach from the dish?

Thanks.
weimin

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Hi Weimin,

yes, you should collect the floating cells as well.

Raffaela

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Hi, Raffaela,

Thanks. after treated with a cytokine or control, nealy all of cells survived well, no cells in suspension. so i do not collect medium only the attached cells for PI. as expected, no apoptosis was observed in both treatments. but interestingly, cells treated with a cytokine has less cells under microscopy inspection than control treatment, but PI staining showed more cells treated with cytokine are in s/G2M phase than controls. Do you have some explanation?

weimin

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Could it be a G2 arrest?

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Yes, it is a possibility. Do you have some idea about confirming this?

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biochemically; G2 checkpoint arrest shows increase of some cell cycle protein such as p16, cyclin B1 and others; it depends I think on your cell cystem...

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I would try witha cyclin

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Hi, All, I will try cycB1 and back to you. but do u think real-time pcr is a good method? thanks

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good idea; moreover, increase of protein expression can be demonstrated with cyclin B1-specific Ab in Western blots; both methods shown would be ideal

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BTW, if cells goes into G2 arrest, will they apopotosis or will they go to differentiation?

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