Utilization of imaging flow cytometry to define intermediates of megakaryopoiesis in vivo and in vitro

•Quantification of rare primary megakaryocytes and their ploidy separate from common contamination by imaging flow cytometry.•Common analytical approach allows comparison of in vivo, in vitro, live and fixed permeabilized megakaryocytes.•General strategies for using the shape, density, texture and coincidence of fluorescence to analyze challenging populations.

Imaging flow cytometry is a particularly powerful analytical approach for the study of megakaryopoiesis. It can utilize well-defined immunophenotypic markers as well as assess maturation of megakaryocytes by their increasing ploidy as they endoreplicate. Imaging flow cytometry can also assess morphometric cell characteristics of size and nuclear to cytoplasmic ratio, which are informative indications of maturation. However, megakaryopoiesis is challenging for flow cytometric analysis, particularly in vivo, because megakaryocytes are very rare in the bone marrow and their odd shape, high DNA content and cell size are similar to clumps of cells. Additionally, both megakaryocytes and immunophenotypically similar platelets are frequently found associated with other cells. Due to these challenges, imaging flow cytometry of megakaryopoiesis exemplifies several strengths of this approach in utilizing fluorescent signal's shape, texture and overlap with other fluorescent signals to distinguish megakaryocytes from a variety of contaminants and to restrict analysis to megakaryocytes, even when associated with other cells. Presented here is a strategy for imaging flow cytometric analysis of rare murine megakaryocytes directly from the bone marrow as well those grown in vitro and analyzed as live cells, or after fixation and permeabilization.