Hello, Dr. Zunino, You have identified the prime difficulty in trying to identify Apoptotic events in a single-parameter histogram. In large measure, Apoptotic events are indistinguishable from debris using PI staining alone. Thus, secondary markers of Apoptosis such as Annexin V are employed for 2-parameter differentiation of "true" Apoptosis. ModFit LT uses the historical assumption of a Apoptotic events appearing as a more-or-less Gaussian distribution sub-G0G1, which is of course in the debris range also. In the absence of a defined distribution over which the Apop range can be placed, the Apop range "competes" with the debris range for events. This allows subtle differences in the placement of the Apop range and the debris (E1) range to effect major changes in the relative results. Since attempts at gating out debris produce their own set of assumptions and problems, assessing Apoptosis in single-parameter PI distributions remains problematic for all DNA analysis software. Please let me know if you may like a further discussion on this topic. Best regards, Mark Mark E. Munson Sales Manager Verity Software House, Inc. Phone: 207-729-6767 Fax: 207-729-5443 verity@vsh.com http://www.vsh.com -----Original Message----- From: Susan Zunino [mailto:szunino@biologie.uni-erlangen.de] Sent: Wednesday, July 21, 1999 8:04 AM To: cyto-inbox Subject: I would like to get some information from anyone who is successfully using the Modfit DNA Analysis program for analysis of DNA content, cell cycle, and especially apoptotic populations. We are having quite a bit of difficulty getting useful information from this program regarding apoptotic populations. We are using mostly PI staining of nuclei for analysis. We are also using cell lines, not clinical samples, so there is limited involvement of an aneuploid population. The main trouble is setting the markers so the data makes sense. We either see an apoptotic population under the G1 and S phases of the histogram or the apoptotic population is labeled as debris. We have no consistency in our data with regard to apoptosis. The manual is not helpful. If anyone out there can help, it sure would be appreciated. Best regards, Susan Zunino Dr. Susan J. Zunino Lehrstuhl fur Genetik University of Erlangen Staudtstr. 5 91058 Erlangen Germany Tele. 49 9131 8528784 FAX 49 9131 8528526 e-mail: szunino@biologie.uni-erlangen.de
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