Multivariate flow cytometric characterization and enrichment of murine megakaryocyte progenitors (CFU-Meg)

Multivariate flow cytometric analysis and sorting of mouse bone marrow cells viably stained with Hoechst 33342 (HO) was utilized to obtain a subpopulation enriched at least 30- to 60-fold for clonogenic megakaryocyte precursors (CFU-Meg). HO fluorescence intensity, forward light scatter, and perpendicular light scatter were measured on each HO-stained bone marrow cell as it passed through a dual beam flow cytometer. Cells were sorted on the basis of their light scattering and HO fluorescence intensity properties. The sorting region which included cells with high forward light scatter, low perpendicular light scatter, and low (0.1-0.65 times the modal channel) HO fluorescence intensity contained approximately 0.6% CFU-Meg. Of all CFU-Meg, 75% were contained in the light scatter gate, and 56% of all CFU-Meg were in the low HO-sorting region. This technique offers a simple, one-step procedure to obtain preparations enriched in murine clonogenic megakaryocyte precursors.