Nathalie Tessier writes- >We have to sort murine megakaryocytes. Can anyone recommend a good >staining and is > Hoescht 33342 staining a good solution to identify that population of >less than 1%. In other words, >can we really discriminate that 6N and more population ???? Would it be >better to enrich the bone marrow by depleting other population by MACS. In the race to isolate megakaryocytic growth factors, Kuter et al developed a flow cytometric assay based on 2-parameter flow cytometric measurement of ploidy and surface antigens using PI and a polyclonal antibody to rat platelets. They used a Cytomutt, triggering on fluorescence with the threshold set above 4N, and a rectangular gate to include antibody-positive events with DNA above 4N. This reliably identified megakaryocytes, even those cells represented under 1% of the nucleated cells in marrow. I would imagine you could use the same tricks for sorting, with an anti-platelet antibody or something more specific and Hoechst 33342 in place of PI - the only problem would be if the cells pumped out the Hoechst 33342, which mouse cells may do, but you might be able to block the pump with trifluoperazine, verapamil, etc. By the way, they got the growth factor at about the same time as several larger and better funded groups, thanks in large part to the ploidy assay. The references are: Kuter DJ, Greenberg SM, Rosenberg RD: Analysis of megakaryocyte ploidy in rat bone marrow cultures. Blood 74:1952-1962, 1989 Kuter DJ, Rosenberg RD: Regulation of megakaryocyte ploidy in vivo in the rat. Blood 75:74-81, 1990 Kuter DJ, Beeler D, Rosenberg RD: The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production. Proc Natl Acad Sci USA 91:11104-11108, 1994 -Howard
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