?Thresholding for ApopTag acquisition

Nan Harvey (nharvey@fiona.umsmed.edu)
Mon, 5 Aug 1996 10:50:50 -0600

I have been acquiring ApopTag-stained samples (FITC) counter-stained with
PI with the threshold set on the Fl2 parameter, as I normally would use for
acquiring cell-cycle data. However, I have a new observation and concern
about the correct thresholding parameter.

Samples with moderate to low percentage apoptosis ran very slowly.
Samples with high percentage apoptosis ran much faster. Could the increase
in flow rate be due to fragments of nuclei being detected by the flow
cytometer?

I set the cytometer so that the G0/G1 peak fell at channel 250 (linear
scale) on the FL2-A plot. For analysis, I gated on the singlets from the
FL2-W vs. FL2-A plot.

Should I be thresholding on FSC instead of FL2? If I threshold on FSC,
where should the cut-off be? Any thoughts on the matter will be
appreciated.

Thanks!

Nan Harvey
University of Mississippi Medical Center
Jackson, MS
nharvey@fiona.umsmed.edu

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