Help for Flow TUNEL analysis
jswigget@mail.carolinas.org
Tue, 13 May 97 08:33:41 PDT
I am running a TUNEL assay (currently without PI) for a researcher that has
preprepared it and brought it to me for analysis (I have some influence over
the manufacturer chosen for the kit, but price tends to govern more than my
input). The researcher is working with chondrocytes grown in a 3-D culture
in agarose. The company has told me (if I understand them correctly - no
cytometrists on board to answer questions) that a neg will have 2 peaks on a
single histogram and a positive will have 3. Currently I am not using any
gate to look at this data, just total 530nm fluor, primarily because on FSC
and SSC; first, I cannot get all possible populations on my oscilloscope at
one time and have had so few cells available I could spare none to look
under a fluorescent scope and second, I can find no information telling me
that I need to use or not use an FSC/SSC or other gate (I'm not sure the
company people understood what I was asking when I asked about gating). So
my first problem is "To gate or not to gate?". My second problem is that
what I do get on FSC/SSC scatter is odd looking; 1 population with very,very
high SSC off-screen, 1 population with very,very high FSC off-screen and a
third population of either what appears to be a debris pattern increasing
primarily in the FSC direction from the origin or a broad FSC spread with
the same SSC throughout (from the origin, on a 2-D plot - a long thin
population). I do realize that free apoptotic bodies are in this equation
and may indeed be reason enough not to use an FSC/SSC gate, but I would like
to hear what others would do in the same situation. My third problem is
that some negatives have only 1 peak and some positives have only 2 and in
many cases the second peak percentage on the positive (with or without a 3rd
peak) increases above the second peak percentage on the negative, from what
I gathered that the company was telling me, I do not bother with second peak
percentage increase only the percent increase in the third peak and that
causes me some concern, esp. when there is no third peak. I would greatly
appreciate any and all input, questions, etc. I will fax any info that
would help to explain my dilemma. You may phone or email me directly at the
phone # or email address listed below. Thanks in advance, Jeanene Swiggett
-------------------------------------
Name: Jeanene Swiggett
Institution: Carolinas Medical Center; Charlotte, N.C.
E-mail: jswigget@carolinas.org
Phone: 704-355-7269
Date: 5/13/97
Time: 8:33:42 AM
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