Re: PI Staining
Russ Boggs (rboggs@earthlink.net)
Wed, 18 Jun 1997 07:05:45 -0800
>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=B0.
After that, I added the RNaseA and PI mixed togother.
--Russ Boggs
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