TdT staining for apoptosis

Ryan Hung (rhung@vcn.bc.ca)
Mon, 11 Aug 1997 12:53:30 -0700 (PDT)

Well, having gotten propidium iodide to finally work, I'm embarking on TdT
staining to assay for apoptosis, as I'm trying to do dual TdT and surface
antibody staining to identify apoptosis in heterogeneous populations of
cells (human PBMC). BTW, thanks to the mailing list for all the help on
PI staining--the secret turned out to be knowing what to look for. To
those who are still having trouble with the technique, make sure you're
using FSC and SSC settings for nuclei rather than for whole cells. I
initially set FSC and SSC parameters using whole cells as control samples,
then when I fed the PI-stained sample through, I got nothing, as they were
nuclei suspensions.

Now, with the TdT assay, I've been running into difficulties determining
where the apoptosis cells actually are. I'm using the Boehringer Mannheim
"In Situ Cell Death Detection Kit, Fluorescein". However, the pamphlet is
not at all clear on how to analyze the data. What I have observed so far
is this: with negative control cells displayed on a logarithmic histogram
of FL-1, I get one large peak at around 10^1. With positive control
(camptothecin-treated) cells, that large peak is shifted slightly to the
left. Furthermore, the number of cells with high dUTP incorporation
increases.

My difficulty in interpretation lies in this: which is the significant
property of the data? The shift in the large peak to the right, or the
increase in high dUTP incorporation? In literature, either one or the
other seems to be used to evaluate degree of apoptosis. Does anybody have
any suggestions about interpretation of data from the TdT assay?

Thanks,
Ryan.

_/ \__/ \__/ \__/ \__/ \__/ \__/ \__/rhung@vcn.bc.ca__/ \__/ \__/
\_Apoptosis=programmed cell death/ \__/ \rwhung@unixg.ubc.ca_/ \__/ \__
_/ --you can't live without it!/ \__/ \http://www.vcn.bc.ca/people/rhung
\__/ \__/ \__/ \__/ \__/ \__/ \__/ \My words Copyright (C) 1997 \__