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Two-color flow cytometric measurement of DNA distributions of rat
megakaryocytes in unfixed, unfractionated marrow cell suspensions
CW Jackson, LK Brown, BC Somerville, SA Lyles and AT Look
The ploidy distribution of megakaryocytes shifts in response to platelet
demand and thus provides a sensitive index of megakaryocytopoiesis. Flow
cytometry (FCM) is a potentially valuable method for rapid determination of
ploidy distributions of megakaryocyte populations; however, because
megakaryocytes constitute only a very small proportion of the cells in
unfractionated marrow, other rare events, such as cell clumping, complicate
FCM analysis. We describe the measurement of cellular DNA distributions of
megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a
method based on the resistance of megakaryocytes to hypotonic lysis in the
cold for at least 2 days. Specific platelet antiserum was used to label
megakaryocytes by indirect immunofluorescence with fluorescein (green
fluorescence), and DNA was stained with propidium iodide (red fluorescence)
in hypotonic citrate solution. The ploidy distribution of megakaryocytes
was selectively determined with two-color, green-gated FCM, with which the
red and green fluorescence of all cells is analyzed, but only the red
fluorescence (DNA content) of cells that specifically bound the platelet
antibody is recorded. We demonstrate that this method can readily detect
changes in megakaryocyte DNA distributions due to experimental
thrombocytopenia or platelet hypertransfusion and, therefore, should be
useful for both experimental and clinical investigations of
megakaryocytopoiesis.
Volume 63,
Issue 4,
pp. 768-778,
04/01/1984
Copyright © 1984 by The American Society of Hematology
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