Released proplatelets mature into multiple individual platelets in vivo. (A) Washed mouse whole blood platelets were run directly on the flow cytometer (negative control) or labeled with 5 µM CMFDA before analysis (CMFDA+ control). The CMFDA+ mouse whole blood platelet population was used to establish forward/side scatter and fluorescent intensity parameters used to identify newly released platelets. Released proplatelets from mouse fetal liver cell culture were labeled with 5 µM CMFDA and transfused into live mice. PRP was collected immediately after transfusion (CMFDA+ <2 min) and 6 h after transfusion (CMFDA+ 6 h), and analyzed by flow cytometry. CMFDA+ platelets were identified using the aforementioned gates. CMFDA-labeled platelet counts were comparable at <2 min after transfusion in CMFDA-labeled platelet and cultured proplatelet transfused mice (≥1% platelet recovery and ≥0.1% of circulating platelets). (B) CMFDA-labeled proplatelets were transfused into mice, and whole blood samples were collected via retro-orbital bleeding at regular intervals over a period of 72 h, commencing with a <2-min time point. PRP was isolated, and platelets were analyzed by flow cytometry and immunofluorescence microscopy. (C) Representative pictures of CMFDA-labeled proplatelets before transfusion and mouse PRP after CMFDA-labeled proplatelet and platelet transfusions. Fluorescence and DIC images were overlaid and merged. Arrows indicate CMFDA-labeled platelets recovered from recipient mice 2 h after transfusion, demonstrating maturation of released proplatelets into multiple individual platelets within the circulatory system. Released PPs were also stained for tubulin (red) and DAPI (blue) to confirm CMFDA labeling of proplatelets before transfusion. CMFDA fluoresces at 458 nm and is shown in green. Immunofluorescence images are exhibited as a montage of representative cells from the same sample slide. Error bars indicate mean ± standard deviation.