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Most flow cytometrist would agree (I think) that events that have a DNA
content of 10% or less than that of the G0/G1 cells are no longer apoptotic
cells. (These events could be apoptotic "bodies" or "fragments" or DNA
fragments or "debris" or ...). One apoptotic cell could have formed several
of these apoptotic bodies, and therefore they should not be counted as
apoptotic cells. Actually, I feel better if the apoptotic cells have better
than 50% of the DNA content of the G0/G1 cells (and the two peaks are well
resolved from one another) to avoid classification arguments.
Set your threshold on FL2 such that you eliminate events below channel 25
(when your G0/G1 peak is in channel 250 on a linear scale), or set the
threshold slightly lower and use a live gate on FL2. If you aren't worried
about missing octaploid or tetraploid cells, consider placing the G0/G1
peak in channel 400 to spread out the hypodiploid events (and threshold/
gate at channel 40).
If you're worried that you're collecting too much junk (and not enough
cells of interest), there are other options. One is to set a count gate on
your G0/G1 peak, and always count, say, 10,000 G0/G1 cells, instead of
counting 10,000 events. Another is to use a time gate, and collect events
over a fixed time period (for example, 2 minutes). You'll end up with
larger list-mode files, but (hopefully) each will have enough cells of
interest.
/\/\/\_ Eric Van Buren, vanburen%flovax.dnet@rocdec.roc.wayne.edu
\ \ \ Karmanos Cancer Institute and Wayne State University,
\_^_/ Immunology & Microbiology, Detroit, Michigan
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