MK culture and PPF from fetal liver. (A) Live-cell images of MKs cultured from fetal liver of A⁺/A⁺, A^gfp/A⁺, and A^gfpR702C/A⁺ embryos. Phase image is shown for A⁺/A⁺ (Ai), fluorescence/DIC overlay image for A^gfp/A⁺ (Aii), and fluorescence z-stack images for A^gfp/A⁺ (Aiii) and A^gfpR702C/A⁺ (Aiv). Arrows indicate proplatelets; arrowheads, platelet buds; green: GFP. Bar, 20μm. (B) Fixed MK cultures stained with anti-CD41 to confirm the identity of the MKs. Note the difference in the length and thickness of the proplatelets (arrows) and the size of the proplatelet buds (arrowheads) between control (Bi-ii) and A^gfpR702C/A⁺ (Biii-iv) MKs. Red indicates CD41; and green, GFP. Bar, 20 μm. (C) Percentage of MKs undergoing PPF in culture (mean ± SD). A⁺/A⁺: 30.8% ± 12.6% (n = 5); and A^gfpR702C/A⁺: 7.4% ± 1.9% (n = 9). *P < .001 (t test). (D) Proplatelet bud size in culture. Measurements of the diameters of proplatelet buds are from the confocal microscope images of live A^gfp/A⁺ and A^gfpR702C/A⁺ MKs. Box plot shows median, upper and lower quartiles, and upper and lower 5 percentiles of each group. A^gfp/A⁺: 3.07 ± 1.05 μm (n = 199); and A^gfpR702C/A⁺: 5.84 ± 2.73 μm (n = 107); *P < .001 (t test). (E) In vitro production of platelets from A⁺/A⁺ and A^gfpR702C/A⁺ embryonic littermate MKs. DIC imaging shows A⁺/A⁺ (Ei) and A^gfpR702C/A⁺ (Eii; GFP signal included) MKs separated from 4-day fetal liver culture by 1.5%-3.0% BSA gradient before PPF; (Eiii-iv) immunofluorescence staining with CD41 Ab to identify platelets after in vitro platelet production by shaking at 150 rpm for 2 hours at 37°. (F) Quantitation of platelet diameter from A⁺/A⁺ and A^gfpR702C/A⁺ in vitro platelet production.