Mouse megakaryocytes differentiation in vitro and flow cytometry analysis of the primary cultures.

(A) Acetylcholinesterase staining of mouse bone marrow cells before (left panel) and after (right panel) in vitro differentiation for 4 days. Arrows indicate the position of mature megakaryocytes, which are easily distinguished from other cells by their large size. Bar indicates 25 μm. (B) Cells cultures characterized by flow cytometry showing dot plots for forward (FSC) and side (SSC) light scatter profiles of 10 000 cells. In each blot, 3 arbitrary analysis gates have been drawn. The lower gate includes small cells; the middle gate, intermediate-size cells; and the upper gate, large cells. Note a progressive increase in the proportion of intermediate-size and large cells with time. (C) In vitro differentiated mouse megakaryocytes were analyzed for calcium increase by single cell digital imaging. Top panel shows elevation of [Ca⁺⁺]_i on convulxin and thrombin stimulation, and table indicates the increase above basal level of calcium. Results are representative of 3 experiments, with 15 cells being measured in each experiment.