TUNEL Scatter Gating.

Steve Perfetto sperfetto at pasteur.hjf.org
Thu Aug 27 08:44:49 EST 1998


Craig,

It really depends on what you might be trying to determine.  If you are
interested in the total apoptotic cell population than it is appropriate to
include all cells as "seen" by the scatter histogram (FS vs. 90L).  In most of
our assays using activated CD4+ T cells one can see the a small percent 5-10% in
the standard scatter gate and a much higher percentage (>50%) in the APO gate. 
Please note these cells are not considered "dead" which is determined by either
EMA or PI for membrane integrity.  Interestingly,  as cells grow out the
percentage does not increase but reaches a equilibrium between the two gates. 
Also I would strongly recommend the use of a positive control by pre-treating
cells with anti-FAS followed by fixation for all TUNEL assay exps.

Stephen P. Perfetto, MS.,MT. (ASCP)
Walter Reed Army Institute of Research
Department of Molecular Diagnostics and Pathogenesis
1600 East Gude Drive
Rockville, MD. 20850


_______________________________________________________________________________
Subject: TUNEL Scatter Gating.
From:    cirwin at adarc.org at Internet_Gateway
Date:    8/24/98  5:11 PM


Hello,

     I would like to find out what everyone thinks is the most appropriate
way to handle scatter gating for the TUNEL assay.
     I am using four colour cytometry to analyse the apoptosis of different
lymphocyte subsets by TUNEL in-vitro.  As far as I undersatnd people
usually include the cells that have small FSC and "more granular than
lymphocyte" SSC in their analyses? These cells are what I usually consider
dead cells.  They also converge with a population that gives beautiful
background if that is what I was looking for.  It's not.  I have seen
publications where people use just SSC as an indicator of apoptosis so is
it OK to set up a gate almost however I chose .
     A second method that I have used is to use a marker like CD3 and
display the data as a histogram of CD3 vs. SSC.  For analysis then I would
gate on the CD3+ cells, but as cells apoptose they decrease their CD3
intensity so the boundry of the gate is very important.
     What do you think is the correct way?  Any Tricks or does it not
really matter as long as all of my results are all consistent?

Thanks for the input,

Craig Irwin

The Aaron Diamond AIDS Research Center
Virology Core





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