Kelley,
Your protocol is essentially on track, but I would like to offer a few
modifications. After typsinization, wash cells once with PBS (or PBS+1%BSA
if you have treated the cells with an agent which may compromise the
integrity of the plasma membrane; the protein will prevent excessive cell
loss). Resuspend pellet in cold PBS, then add cold EtOH while vortexing.
This will help prevent cell clumping. My final EtOH concentration is 70%.
I store my cells at -20 C for up to 2 weeks, but I have read where others
store cells at room temp. After pelleting cells, I resuspend in PBS
containing 200 ug/ml RNaseA and 5 ug/ml PI, then incubate at room temp in
the dark for 30 min. I would not recommend freezing the cells after
staining. If you are unable to analyze them the day you stain them, leave
them in the EtOH and stain them when you are able to analyze.
I think you will find mammalian cells are a breeze to stain compare to
yeast! Good luck!
Lisa Adams
Pharmacia & Upjohn
LAADAMS@am.pnu.com
______________________________ Reply Separator _________________________________
Subject: PI Staining
Author: Kelley Lennon <klennon@bu.edu> at INTERNET
Date: 6/17/97 2:24 PM
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
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