Can't see G2/M peak with PI staining

[rtruitt3]

I'm trying to stain colon cancer cells with PI to see both G0/G1 peak and G/M peak. I can see the G0/G1 peak, but it's not as sharp as I think it should be and I can't even see the G2/M peak. My method is the following:

Collect cells with trypsin, aspirate media from cell pellet
Treat cells with staining solution that contains 5microgram/mL PI, 5 microgram/mL boiled RNAse A in PBS, sodium citrate (i forget concentration), and 0.1% Triton X-100.

I incubate cells with staining solution in dark at 37C for 10min
Then I add NaCl to solution (i forget how much, don't have protocol with me)

Any suggestions would be helpful, I'm really just want this to work.

Thanks

[pymzz]

Hi

I work with skin fibroblasts. After trypsisation I wash with PBS and then fix cells in 70% ethanol and leave at -20 degrees C at least overnight. Then I add PBS, pellet cells, wash again with PBS and resuspend in PBS with 50 ug/ml PI and 20 ug/ml RNAse and leave them overnight at +4 degrees C.

It sounds like you used the Nicoletti buffer, without the 70% ethanol fixation (Journal of Immunologwal Methods, 139 (1991) 271-279). This is suitable for measuring sub-G1 cell debris (often used as a proxy measure for apoptosis) but less for G1-S-G2 analysis.

Hope this helps