Kinetics of preplatelet maturation. (A) Whole blood and washed platelets anticoagulated with trisodium citrate (citrate blood) was incubated for 3 hours at 37°C to determine change in barbell formation quantified by ISFC at 0, 1.5, and 3 hours and labeled with FITC anti-CD61 and AF674 SiR tubulin (4 μM; n = 6). Washed platelet barbell perimeter measured by ISFC (n = 6). SiR tubulin live-cell labeling of washed platelet barbells (n = 3; bar represents 7 μm). (B-C) Experiments were conducted with washed platelets from human control citrate blood incubated in M199 medium at 37°C for a maximum of 24 hours. (B) Barbell platelet formation at 0, 1.5, 3, 6, and 24 hour time points (visualized using the image flow cytometry barbell gate described in supplemental Figure 3). Quantification of preplatelets and barbells at 0, 1.5, 3, 6, and 24 hour time points (n = 10). (C) before incubation, washed platelets were labeled with either CellTrace™ green (0.2 μg/mL) or red (1 μg/mL) cytosolic dyes, mixed, and incubated for 3 hours to demonstrate that barbells originate from single platelets (n = 3; imaged by ISFC, ×60 magnification; bars represent 7 μm). A*Maj-Ax-Int, area × major axis intensity; Min-Ax-Int, minor axis intensity. (A-B) Two-way analysis of variance with Bonferroni multiple-comparisons test ±1 standard deviation. *P < .05, **P < .01, ***P < .001.