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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
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web