MKs derived from ES cells deficient in WAVE2. (A) Immunoblots of WAVE2, WAVE1, Abi1, and tubulin from lysates of day-5 embryoid bodies (EBs). To avoid contamination with mouse feeder fibroblasts, day-0 ES cells, were sorted with c-Kit⁺ cells, applied to EB media,³³  and cultured for 5 days. (B) Flow cytometric dotplots of side and forward scatter profiles of 10 000 live cells on day 8, and expression profiles of α_IIb and GPIbα stained cells (day 8), as described previously.^28,29  On day 12, all cells collected from a 6-well culture plate were stained with anti-α_IIb, anti-GPIbα, and propidium iodide and were mixed with True Count Beads. WAVE2^+/+, WAVE2^+/−, or WAVE2^−/− cells and beads were then subjected to flow cytometry. Representative dot plots show all viable cells from WAVE2^+/+ and WAVE2^−/− plates. Quantification for GPIbα⁺ cells in an arbitrary gate was performed as described in “Materials and methods.” The graph on the right shows means (± SD; n = 3). (C) On day 8 or day 12, weakly adherent or floating cells^28,29  were subjected to cytospin preparation and to staining by Wright-Giemsa technique. WAVE2^−/− MKs showed decreased number of large sized cells. (D) On day 12, live cells were fixed following cytospin preparation. Cells were permeabilized, stained with anti-VWF followed by rabbit Alexa 488 (green), and anti-α_IIb followed by mouse Alexa 568 (red) with nuclei marked by 4,6-diamino-2-phenylindole (DAPI) staining (blue). WAVE2^−/− MK retained VWF expression (diffuse but decreased) in cytoplasm while VWF expression in WAVE2^+/+ MK was distributed clearly along the plasma membrane in peripheral cytoplasm (◀). (E) On day 11 (i) or day 12 (ii), proplatelet-exhibiting MKs derived from normal ES cells were fixed and plated on poly-L-lysine–coated cover glasses. Cells were permeabilized and were stained with rhodamine-conjugated phalloidin to mark F-actin (red) and with anti-WAVE2 antibody followed by Alexa 488 (green). Arrowheads indicate swellings. See “Image acquisition for platelet and MK spreading” for complete image acquisition information. (F) The left figure shows means (± SD; n = 3) of total number of proplatelets in a 6-well plate on day 12. The right figure shows means (± SD; n = 3) of number of α_IIb⁺ particles within the same flow cytometry gate as that used for murine platelets. Quantification was performed using True Count Beads as described in “Quantification of mature MKs and platelets.”