The gating strategy for sub-G1 cells is described in detail in Meth Cell Biol.,Vol 63: pp 533-4, 2001 and also in the review in Cytometry, 27, 1-20, 1997. Unfortunately, many authors that use this methodology, use it erronously. In short, these are the critical points. 1. One should not lyse unfixed cells using detergents or hypotonic solutions. Because nucleus of apoptotic cell is fragmented, during cell lysis many nuclear fragments are released from a single cell. Most authors count these fragments as individual apoptotic sub G1 cells. This overestimates apoptotic index (AI) many-fold.In addition, lysis of mitotic cells releases individual chromosomes or chromosome aggregates - each of them is erronously counted as an apoptotic cell. If the cells were treated with taxol or other mitotic poisons, this can result in incredibly high error !!!! Micronuclei also are released and also are erronously counted as apoptotic cells. 2. One should not use logarithmic scale for ploting intensity of DNA-associated fluoprescence. Note that when using log scale many authors identify and count the objects with DNA content less than 1 % ,and some authors with less than 0.1% of that of the whole G1 cells, as apoptotic "sub G1" cells. The objects with so little DNA content cannot be the whole apoptotic cells. Most likely they are small chromatin (nuclear) fragments or other objects discussed in point #1. 3. I suggest that the cells have to be fixed in ethanol and then extensively rinsed, pehaps even with high molarity buffer to extract fragmented DNA, prior to staining. Then the sub-G1 cell fraction should be identified (counted) by setting the low level gate at 5 or 10 % value of the G1 peak. Thus, if the peak of G1 cells is at channel 200, the objects with fluorescence lesser than 10 or 20 channels should be excluded. The 5 or 10 % level is, of course, arbitratry and may lead to understimation of apoptotic cells. The degree of underestimate, however, is consistent and reproducible from sample to sample. Zbigniew Darzynkiewicz, M.D., Ph.D. New York Medical College 19 Bradhurst Ave. Hawthorne, NY 10532 tel: 914-347-2801 fax: 914-347-2804 http://www.geocities.com/z_darzynkiewicz > -----Original Message----- > From: Inna.Cohen@med.va.gov [SMTP:Inna.Cohen@med.va.gov] > Sent: Monday, December 02, 2002 2:50 PM > To: Cytometry Mailing List > Subject: Gating > > > Hello All! > We at the VAMC are having problems deciding where to gate to see a subG1 > peak when we look for apoptosis in the cell cycle. Any helpful > suggestions? > > Thanx, > Inna Cohen > VA Medical Center
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