Just to throw in my two cents, on avian bursa fabrisius (sp?) we have gotten good results w/ PI (the hypodiploid peak discussed) and with the Tunel assay, and will soon be confirming with microscopy. If your apoptotic peak is too low (close to the origin) you might want to reduce the amount of time that the cells are held before analysis; I seem to remember one of the review articles (Dr. Darzynkiewicz et al?) mentioning the position of the peak being dependent on the amount of time fragments are "allowed" to leak out of the cells. Just another suggestion, Steve ------------------------------------------------------------- Steve G. Hilliard Linux: You're in a maze Cell Analysis Facility of twisty packages, University of Georgia all alike..... ------------------------------------------------------------- ---------- Forwarded message ---------- Date: Tue, 3 Jun 1997 16:54:23 -0400 (EDT) From: William George Telford <apoptosi@umich.edu> To: cyto-inbox Subject: Re: FTIC + PI (fwd) My messages keep getting bounced back from your e-mail, so I'm responding via the list. Maybe others will find this useful too... Bill ---------- Forwarded message ---------- Date: Tue, 3 Jun 1997 16:50:01 -0400 (EDT) From: William George Telford <apoptosi@umich.edu> To: cyto-inbox Subject: Re: FTIC + PI Hi Lyndal... Sorry I didn't respond sooner but I've been out of town. One of the major pitfalls of using single dye methods with fixed cells to detect apoptosis is that you often don't get a nice, clean apoptotic peak. The state of the apoptotic cells with respect to DNA content after DNA fragmentation is quite variable depending on the cell type. We were fortunate to initially try the method with mouse lymphoid cells, which give a nice peak with a relative index of about 0.5 with respect to the G0/G1 diploid peak. We later found that cycling mouse lymphocytes and human cells often gave a peak well below this, often at the threshold of instrument sensitivity on the linear scale. Some people have tried to use log scaling to quantify these very dim apoptotic cells, but they are often not very clearly defined even then. If you are seeing this type of apoptotic peak (with the cells smeared up at the minimum linear threshold), then you might be measuring both apoptotic cells and cellular debris, giving you an exaggerated number of apparent apoptotic events. So, what to do? Try analyzing the cells on a log scale; this might allow you to detect multiple subdiploid populations, one or ore of which should be apoptotic. You can also analyze your cells for both DNA fluorescence and forward scatter, and then exclude events with overly low FS signal; this will allow you to separate apoptotic cells from debris, etc. that have the same apparent DNA fluorescence. You might also want to try Dr. Darzynkiewicz's protocol where he uses phosphate/citrate buffers of different concentrations during the DNA staining process in an attempt to control the amount of DNA loss following fixation. I don't have the references with me (I'm still out of town!) but I can give them to you once I get back. The other option is to try another apoptosis detection method! Single dye detection methods have sort of "fallen into disfavor" recently for the variability of the apoptotic regions depending on cell type. We still use them but recognize their limitations (fortunately,most of my work is with mouse lymphocytes, where they work quite well ;-). You might want to independeently confirm your results with a different method (two dye membrane exclusion, FITC-annexin, etc.). It is also a good idea (although a pain) to sort the cells you think are apoptotic and check them for DNA fragmentation by electrophoresis, or morphology by confocal or EM. If you want, send or FAX me a copy of the histograms you are getting, and I can compare them to what we've seen before. Our address here is: Hospital for Special Surgery Flow Cytometry Core Facility Research Building Room 312 535 E. 70th Street New York, NY 10021 Our phone and FAX numbers are: phone: (212) 606-1925 FAX: (212) 717-1192 Good luck! Let me know how things go. Cheers! Bill
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