>I am staining cultured cells (CHO) with propidium iodide for FACScan >analyses. I have stained yeast tons of times with PI, but this is my first >attempt with mammalian cells. I am wondering what I should do differently. >I realize that I can't sonify my CHO cells like I do my yeast, but I guess >what I'm wondering is how careful I have to be with them. My protocol is >as follows: > 1) trypsinize cells, pellet, resuspend in 1 ml PBS and 4 mls >absolute ethanol; store at -20C > 2) pellet cells, resuspend in 1 ml PBS; add 100 ul RNAse A (200 >ug/ml), incubate at 37C for 30 min > 3) add 100 ul 1 mg/ml PI; let sit at room temp 5-10 min > >How gentle do I have to be with them when I'm resuspending them? Can I do >the staining and then freeze them at -20C until I want to analyze them? > >Advice would be greatly appreciated (I'd hate to have a big mass of < 2N >cells because I lysed them!!!!! What worked for me was to pellet the cells as in step one, but then add dropwise (P1000) one ml of ice-cold 70% ethanol while vortexing. I can't remember if I vortexed at half-speed, but the cells were definitely continuously vortexed. Stored at -20°. After that, I added the RNaseA and PI mixed togother. --Russ Boggs
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