TUNEL Scatter Gating.

Howard Shapiro hms at shapirolab.com
Tue Aug 25 16:39:56 EST 1998


Craig Irwin asks:
>
>     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?
>

Gating is THE trick in flow cytometry, but this isn't an easy gating
problem.  The apoptotic cells typically have lower FSC and higher SSC than
the nonapoptotic cells, so a typical "tight" lymphocyte gate will exclude
most of the apoptotic cells, and is inappropriate.  If interference from
"dead" (i.e., necrotic) cells becomes a problem, then one has to consider a
way of discriminating viable, necrotic, and apoptotic cells; this can be
done based on different permeability to various nucleic acid dyes if the
cells are not fixed, but gets much more complicated if they are.  And, yes,
if the CD3 intensity decreases during apoptosis, gating on CD3 vs. side
scatter can become impossible.  There aren't any easy answers to the
question, and consistency is probably the best thing to aim for.

Under many circumstances, many of the "dead" lymphocytes will have become
necrotic after first undergoing apoptosis, so, if there isn't some
alternative pathway leading to necrosis of a significant fraction of the
cells, a gating strategy which mixes some or all of these cells with the
apoptotic cells may be acceptable.  In theory, the TUNEL assay itself could
discriminate the two classes of cells.

In our work with stimulated lymphocytes using a dual-beam (UV/488)
instrument and staining for DNA with Hoechst 33342, my colleagues and I find
that low Hoechst dye fluorescence is a pretty good indicator of apoptosis;
unfortunately, it's very difficult to obtain a DNA measurement with a good
CV in a four-color system using 488 nm or 488 and 633 nm excitation, which
makes it hard for most people to play this game.

-Howard




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