re:apop and flow
Robert Johnson (Marcia.Woda@BANYAN.UMMED.EDU)
Wed, 3 Jan 96 13:27:15 EST
Before I get to the subject of apoptosis, I want to say that I am in favor of
keeping this mailing list as it presently is run receiving messages almost in
real time as someone else pointed out, and no censorship or compiling of
messages. One can quickly delete messages that are of no interest and print
out those to save for future reference. There is so much to be learned and
information to be shared.
We here perform several different flow assays in order to count
apoptotic cells. With any assay a threshold on some parameter must be set so
that the machine knows when to count, and we tend to set this at the lowest
possible setting so that we see everything in the list mode file when we go
onto analysis. It was very useful to read Dr. Darzynkiewicz's posting just
recently, and I hope I am paraphrasing it correctly, that in the assay in
which cells are fixed, low mw DNA extracted and cellular DNA measured with
lin amplifiers, particles with 5-10% ofintact G1 cells represent apototic
bodies, not cells. In this assay we set our G1 peak at channel 400 (1024
channels) and events below channel 30-40 correlate well with results of
agarose gels on sample aliquots.
On live cell assays with Ho33342/PI or with the tunel assay it is even
more difficult to know what is truly debris by PI staining or light scatter.
Again we set a very low threshold on the PI channel or FSC respectively and
try to make sense of the data offline. Cells we are studying undergoing
apoptosis do not look like the classical thymocyte picutures, and end-label
specific fluorescense backgates to cells which appear viable by light
scatter,cells with low forward and side scatter, and more worrisome particles
with even lower fwd scatter. The lowest fwd scatter pop. we interpret as
debris with high background staining. The pop with low forward and side
scatter does not stain 100% pos for end-label which is somewhat of a mystery
but we interpret as some problem with inaccesability to the reagents. The
Ho/PI assay we perform with live cells in order to descriminate necrotic from
apototic is highly reproducible in our hands yet with the PI threshold set
very low there is always a small pop unaccounted for with PI staining
somewhat less than necrotic cells and no Hoechst uptake at all. Perhaps
someone has a different interpretation of these scenario or has suggestions.
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
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If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
