Re: Gating for apoptosis studies
From: William Telford (TelfordW@mail.nih.gov)
Date: Thu Sep 05 2002 - 00:40:40 EST
Hi Dan...
When you do cell cycle analysis by DNA area versus width, generally the
singlet apoptotic cells fall in the hypodiploid region with reduced area
AND width - that high-width subpopulation you see is probably aggregates
of dead cells and debris. However, your hypodiploid region also
probably contains some necrotic cells, debris, membrane blebs, etc. in
addition to apoptotic cells - and since you are at the low threshold of
instrument sensitivity, you can't tell them apart by DNA content analysis
alone (hence the "smear").
I think the general consensus (both from the methods literature and from
discussions on this list) is that it is very risky to attempt to quantify
apoptosis by hypodiploid peak analysis alone, except for very select cell
types such as resting rodent immune cells, where the apoptotic peak is
sharp and well-defined from the debris. I think it is OK to
qualitatively determine that you MIGHT have apoptosis if you see a
hypodiploid peak, but should then use a more specific assay (preferably
more than one) to actually quantify these cells. These are a
variety of good cell death assays for flow out there now (TUNEL, annexin
V, fluorogenic caspase substrates, antibodies against active caspase,
etc.), so there are lots of options.
We like to assay for multiple cell death characteristics, combining
assays that look at both earlier and later cell death events.
Combined annexin V binding and DNA dye exclusion (PI or 7-AAD) works well
for unfixed cells - even better are one of several fluorogenic caspase
substrates now on the market, also with a DNA dye. For fixed cells,
you can simultaneously label for TUNEL and immunolabel for active
caspase. You can get a more comprehensive view of the cell death
progression by seeing several stages in a multipronged assay. The
standard apoptosis assay in my lab uses 7-aminoactinomycin D exclusion,
annexin V binding and caspase 3 activity simultaneously for a
multifaceted view of the death process. It is referenced in Telford
et al., Cytometry 47:81-88 (2002), and is also reviewed (along with
protocols) on our website at
http://home.ncifcrf.gov/ccr/flowcore/index.htm
(click on the Projects button and go to the cell death page).
Hope this helps.
Bill Telford
NCI-NIH
At 09:02 AM 9/4/2002 -0400, Rosson, Dan wrote:
Dear Fellow Flowers:
I'd like to ask my more learned flowlosopher colleagues a question on gating for the purpose of quantitating sub G1 content for apoptosis studies. I'm hoping Powerpoint illustrations can be attached to these mass mailings. We all know that we should gate out doublets and triplets in order to restrict a cell cycle analysis to single cells and one of the ways of doing this is to plot Area vs Width in order to draw a region. The Powerpoint attachment is a dot plot of such an experiment. Notice the long trail of sub G1 events that are big, fat and wide. Should these events be gated out as in the first histogram or left in as in the second histogram? It seems to me that if they are two or more sub G1 events are stuck together they're still apoptotic and should be left in the gate otherwise it's cheating the apoptotic population of its due representation. However, I've never seen a discussion of this. So, if some of you have any insight into this, I'd like to hear it.
Sincerely,
Dan Rosson
Dan Rosson Ph.D.
Lankenau Institute of Medical Research
100 Lancaster Ave.
Wynnewood, PA 19096
www.limr.org.
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