Re: megakaryocytes
Howard Shapiro (hms@shapirolab.com)
Sat, 20 Jul 1996 21:18:35 -0400 (EDT)
>From about 1987 to the early 1990's, Dave Kuter, working with Bob Rosenberg
at MIT and using a Cytomutt with a 5 mW argon laser, developed and used a
megakaryocyte ploidy assay to aid in purifying platelet growth factor.
Cultured marrow cells were indirectly labeled with a polyclonal
anti-platelet antibody/fluorescein developing antibody and propidium iodide;
PI fluorescence was used as the trigger signal with the level set so that
diploid and tetraploid cells were ignored, and a rectangular acquisition
gate was set to include cells with high (8n or more) PI fluorescence and
high fluorescein fluorescence. Megakaryocytes accounted for approximately 1
in 1,000 nucleated cells in the samples, which were run at approximately
5,000 cells/sec (remember, most of the nucleated cells didn't trigger the
system). This allowed a histogram including 1,000 of the 8n to 64n
megakaryocytes to be accumulated in a few minutes.
(Kuter DJ, Greenberg SM, Rosenberg RD: Analysis of megakaryocyte ploidy in
rat bone marrow cultures. Blood 74:1952-62, 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, 91:11104-8, 1994)
Assuming you had the nozzle hemodynamic problems solved, you could
implement a similar solution for viable sorting using Hoechst 33342 and
perhaps something more elegant in the way of antibodies.
-Howard