Derek/Mireia/MIchael/Ian: Let me add my two cents to this thread. While I agree that there seem to be some broken cells that don't stain as brightly as you would expect with annexin, I might suggest a second possiblity. Three background facts/assumptions: First, some models show a PI (or EB or 7-AAD) intermediate apoptotic population. Second, this could be due to a mechanism similar to that which causes apoptotic cells to be intermediate for fluorescein diacetate (as originally reported by M Ormerod I think). Third, I have reported (Cytometry 28:253) that in models I look at the surface staining with annexin follows the change in FDA staining. Given the above it would seem possible that a PI-intermediate, annexin-negative population could develop before the double positives. A time course or a careful look at the PI intensity might give you a hint about whether this is the case. Tom Frey _______________________________________________________________________________ Subject: Re: annexin/PI From: Derek Davies <daviesd2@icrf.icnet.uk> at INTERNET Date: 1/28/99 5:23 PM Hello Mireia, I don't think that you are the only one to have come across this problem which does seem to get largely ignored. I suspect that most people are interested in the early apoptotic population (Annexin+/PI-) and just lump together all the PI+ cells together into a "late apoptotic/necrotic" population. This is fine I think until you are called upon to define the Annexin-/PI+ population. At this stage you should bear in mind one of the Flow Commandments: "Stray ye not too far from a fluorescence microscope". You could look at your cell prep (or even sort the relevant populations onto a slide) and see what is what. Hazarding a guess, these cells could be very late dead cells that have lost a large part of their membranes. Derek On Tue, 26 Jan 1999, Mireia Dalmau (S. Radioisotops Bellv.) wrote: > We have been analyzing apoptosis cell death in a human colon > adenocarcinoma cell line, HT29. We have obtained very nice and clear > results, but we usually detect a 3 to 5% annexin (-)/ PI (+) > population. > We can observe the same result with other cell lines and different > stimuli, and I have been asked to explain what is happening > there. > Many articles don't make any difference between PI (+) cells, beeing > annexin (+) or (-). Could someone suggest how to describe these cells ? > > Mireia Dalmau > Servei de Radioisotops i Citometria de Flux > Campus de Bellvitge, Universitat de Barcelona. > C/ Feixa Llarga s/n. 08907 L'Hospitalet > > Telf. 93 402 42 06 FAX. 93 402 42 13 > e-mail: serv-rad-bell@bell.ub.es > __________________________________________________________ > **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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