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Incomplete resolution of G1 -S and G2/M phase after PI staining - cell cycle analysis (Aug/16/2009 )

Hi all,
I am using epithelial cells. After inducing DNA damage, I fix the cells with 2%PFA at RT for 10 mins,followed by permeabilization with methanol. I stain my cells for a nuclear phos pho antibodya nd counter stain with PI for cell cycle. This procedure has worked for several cell types. In epithelial cells although I get a good FL1 signal, There is poor/ no resolution of G1/ S and G2/M phase. A defined S/G2 peak is absent. Instead What I see is a broad G1 peak with a should slant and no G2. Could anyone help me resolve the cell cycle in theses cells....


I'm a little confused by your description. Usually PI staining gives a peak for G1/G0, a second peak for G2/M, and a broad signal between them representing S phase cells. This is because PI stains DNA. G1 and G0 cells have one copy of their DNA, G2/M cells have 2 copies (thus a higher signal than G1/G0), and cells in S phase are busy replicating their DNA so will have somewhere between one and two copies. So I'm very unclear what you mean by resolution of G1/S and a defined S/G2 peak.