Fluorescence cytophotometric analysis of megakaryocytic ploidy in culture:
studies of normal and thrombocytopenic mice
C Chatelain and SA Burstein
A system for the accurate and rapid measurement of the ploidy of cultured
megakaryocytes derived from megakaryocytic colony-forming cells (CFU-M) has
been developed. Thirty thousand murine marrow cells per milliliter were
cultured for varying time periods in agar in the presence of horse serum
and pokeweed mitogen-stimulated spleen cell- conditioned medium (PWM-SCM).
To ensure the inclusion of all the megakaryocytic cells in the analysis,
entire agar discs were transferred onto glass slides and dried. Cells of
the megakaryocytic lineage were identified by staining for
acetylcholinesterase (AchE) for two hours. Subsequently, the nuclei of the
cells were stained using 1.7 X 10(-5) mol/L chromomycin A3, a specific
DNA-binding fluorochrome. Megakaryocytic colonies (greater than or equal to
2 AchE+ cells) were located under transmission light. The fluorescence
emission of each cell of the colony was then measured by a photometer
interfaced with a computer. The mean fluorescence emission of about 20
random granulocytes per slide was used as a 2N standard. There was no
significant cell loss, quenching of fluorescence by AchE staining, or
overlapping of colonies or cells. Approximately 100 megakaryocytes per hour
could be analyzed. Modal ploidy of cultured megakaryocytes increased from
2N to 32N between days 3 and 6 in culture. Varying concentrations of
PWM-SCM from 5% to 20% did not affect the ploidy distribution when examined
at day 5. The heterogeneity of the ploidy of cells within colonies
increased continuously with increasing cell numbers per colony. Clonal
analyses of mean ploidy and ploidy heterogeneity did not show distinct
types or classes of colonies; rather, the data show that megakaryocytic
colonies are structured as a continuum. An inverse correlation was found
between the number of cells constituting the colonies and their mean DNA
content. To determine if short-term in vivo exposure of CFU-M to a
thrombocytopenic environment could affect the ploidy of their progeny, mice
were given rabbit antimouse-platelet serum while control animals were given
normal rabbit serum. Twenty-four hours after injection, marrow derived from
these animals was cultured. At day 5, the ploidy distributions and ploidy
heterogeneity were identical in both treated and control groups. Thus,
factor(s) that promote CFU-M proliferation do not affect megakaryocytic
endoreduplication, while stimuli that acutely influence megakaryocytic
ploidy in vivo do not determine the ultimate ploidy potential of
megakaryocytes derived from a CFU-M.
Volume 64,
Issue 6,
pp. 1193-1199,
12/01/1984
Copyright © 1984 by The American Society of Hematology
