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