From: David Coder (d_coder@MSN.com)
Date: Tue Oct 28 2003 - 13:30:30 EST
Dear María, I would be negligent not to mention that such can be done easily and rapidly with the imaging flow cytometer in development by Amnis Corp. in Seattle. (For the record, I am a consultant to Amnis.) The attached apoptosis summary shows images of Jurkat cells that are viable (Annexin V neg., 7-AAD-neg), early apoptotic (Annexin V pos., 7-AAD-neg), late apoptotic (Annexin-V pos, 7-AAD-pos), or necrotic (positive for all markers. (Apoptosis was induced by camptothecin, and necrosis was induced by hydrogen peroxide. PE-labeled anti-HLA-ABC was used to label class-I only in peroxide-treated cells thus demonstrating penetration by large molecules. Cells from both treatments were mixed for analysis in flow imaging.) Six images for each of 4,500 cells were collected in about 45 seconds. It is quite easy to see the apoptotic bodies in the late necrotic cells and the bodies correlate with antibody labeling patterns. Dot plots of fluorescence intensity of Annexin-V vs. 7-AAD provides the standard plot for examination of viable, apoptotic and necrotic populations. But one cannot discern the transitions from early apoptotic to late apoptotic, to necrotic phenotypes. But when examining the images of the cells collected in flow, derived morphometric parameters (from features computed automatically at the start of analysis) make it easy to distinguish each of the phenotypes (see plot). One can examine the images of each cell in each phenotype simply by clicking on a dot in the bivariate display. Dave ================ David M Coder, Ph.D. Consultant in Cytometry Seattle WA Cell/Msg: 206 499 3446 Email: d_coder@msn.com -----Original Message----- From: Mª Belén Vidriales [mailto:mbvidri@usal.es] Sent: Sunday, October 26, 2003 11:52 PM To: cyto-inbox Subject: Apoptotic bodies Hello all, Has anyone experience in measuring apoptotic bodies by flow cytometry? Protocols and references greatly appreciated. María-Belén Vidriales Vicente Hematology Department University Hospital Paseo de San Vicente 58-182 37007 Salamanca. Spain Tf 34 923 29 13 75 Fax 34 923 29 46 24
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