PI vs. 7-AAD Nuclear Staining

Dennis_Young@CIS.ucsd.edu Dennis_Young at CIS.ucsd.edu
Fri Mar 15 19:26:00 EST 1996

The main problem is that FITC is detected in FL2 AND FL3 and you can't 
compensate FL1 vs FL3 directly. (You can with Verity Software House's WinList, 
but I don't have this!) You can adjust the compensation through FL2 by using 
all compensation settings.
I use the FACStar+ where I can put a better filter in FL3, 630 nM bandpass, 
instead of the 650 nM longpass that is in our FACScan.

Are you setting the compensation values with SINGLE stained samples?

And yes, we all have seen the dim PI positives. I use as litte PI as possible 
(0.5 micrograms per ml) but still have seen them. I've heard some people WASH 
after PI staining, but the A-V protocols are supposed to be no wash!

______________________________ Reply Separator _________________________________
Subject: PI vs. 7-AAD Nuclear Staining
Author:  DJS at allp.com at @UCSD
Date:    3/15/96 10:24 AM

I have been trying to surface stain apopotic MCF-7 breast cancer cells with
Annexin V-FITC and stain the DNA with PI to test for necrosis vs apoptosis.
 Even though the emission spectra shouldn't overlap I am having to
compensate for what appears to be overlap in both FL1 and FL3.
Also the PI appears to be passing through the membrane of viable cells.
Any suggestions on either problem (Compensation or viability stain)
Would switching to 7-AAD be a possibility.
Thanks for the input,
Dan Smith
San Diego
djs at allp.com

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