Mapping preplatelet maturation in vitro. Washed platelets were labeled with 2 μg/mL of CellTrace™ yellow cytosolic dye and incubated in serum-free M199 medium for 24 hours. (A) Scatterplots demonstrate the appearance of a discrete population of platelets termed “progeny,” which display a decrease in CellTrace™ yellow MFI (images depicted by CD61 and CellTrace™ fluorescence, using ISFC, ×60 magnification). Percentage increase in platelet count; the CellTrace™ fluorescence profile at 0 and 24 hours is also demonstrated. (B) AF599 Mitotracker Ros CMX MFI of platelets and platelet progeny with representative images and the effect of rotenone (3 μg/mL) on barbell formation when incubating washed platelets for 6 hours. (C) Washed platelets were incubated for 6 hours with nocodazole (10 μM) or cytochalasin D (1 or 0.1 μM), and barbells were quantified by ISFC. To show the effect of either cytoskeletal drug, marginal band morphology was depicted by using AF674 SiR tubulin live-cell labeling (n = 3). (A-C) Bars represent 7 μm. (A) Paired t-test, (B) Mann-Whitney U test and Wilcoxon test, and (C) 2-way analysis of variance. (A-C) ±1 standard deviation. *P < .05; **P < .01; ***P ≤ .001.