Hi. 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!!!!! Kelley BU Med Center
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:51 EST