There really is no secret about the Hoechst 33342 (or 33258) assay for apoptotic cells. It has been described in several papers. Ormerod MG, Sun X-M, Snowden RT, Davies R, Fearnhead H, Cohen GM. Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. Cytometry 1993; 14 595-602. Dive C, Gregory CD, Phipps, DJ, Evans DI, Milner, AE, Wyllie AH. Analysis and discrimination of necrosis and apotosis (programmed cell death) by flow cytometry. Biochem. Biophys. Acta 1992; 1133: 275-285. Ormerod MG, Collins MKL, Rodriguez-Tarduchy G, Robertson D. Apoptosis in interleukin-3-dependent haemopoetic cells. Quantification by two flow cytometric methods. J. Immunol. Meth. 1992; 153: 57-65. Sun X-M, Snowden RT, Skilleter DN, Dinsdale D, Ormerod MG, Cohen GM. Flow cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Anal. Biochem. 1992; 204: 351-356. Ormerod MG, Sun X-M, Brown D, Snowden RT, Cohen GM. Quantification of apoptosis and necrosis by flow cytometry. Acta Oncologica 1993; 32: 417-424. Elstein KH, Zucker RM. Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic subpopulations. Exp. Cell Res. 1994; 211: 322-331. If anyone is interested to see typical data, there are some listed data files illustrating the method on my CD-ROM - Data Analysis in Flow Cytometry - a Practical Approach. CD-ROM publ. by M. G. Ormerod, 1996. The problem with the method is that it is not universal. It works well with some cells and cell lines, not woth others. Michael Ormerod 34 Wray Park Road Reigate RH2 ODE Telephone: voice & FAX: (0)1737 241726 Web site: http://ourworld.compuserve.com/homepages/Michael_Ormerod
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