-----Original Message-----
From: Rosson, Dan [mailto:RossonD@MLHS.ORG]
Sent: Wednesday, September 04, 2002 9:03 AM
To: cyto-inbox
Subject: Gating for apoptosis studies
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.
Hi Dan,
I have some suggestions.
1] First, I would run a proliferation control, so you can determine the
correct gate for proliferating singlets. The advantage of using
proliferating cells here is that resting cells just provide a single
cluster, whereas dividing cell give you the line on which singlets fall.
Apoptotic cells should fall on the same line.
2] Next, I would run an apoptosis control. Use a positive inducer of
apoptosis for your cell type to determine the position of the apoptotic
cells.
YOu can find more info on using Jurkat cells as apoptotic control cells in
Flow Cytometric Detection and Quantification of Apoptotic Cells, Mc Closkey
and Pahwa, Manual of Clinical Laboratory Immunology, p. 274-283, 6th
edition.
In general, we do see doublets and clumps of apoptotic PBMC, under the
fluorescent scope and on the cytometer. Sinc e one of the assumptions of
this analysis is a gate on single cell events, we gate them out. For PBMC,
we gate on light scatter and a doublet discrimination gate. The machine is
triggered on fluorescence equal to 25% of the G0 peak, the rationale is that
this will exclude confounding events which would erroneaosly elevate the %
apoptosis.
I would also recommend: Cytometry in Cell Necrobiology: Analysis of
Apoptosis and Accidental Cell Death, Darzynkiewicz, Juan, Li, Gorczyca,
Murakami, and Traganos, Cytomtry, 27:1-20, 1997 for further discussion of
some of these issues.
Good luck,
Tom
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Thomas W. Mc Closkey, Ph. D.
Director of Flow Cytometry, North Shore University Hospital
Assistant Professor of Pediatrics, New York University School of Medicine
Boas Marks Biomedical Research Center, 350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866
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