Activated platelets release MPs that contain miR-223. (A-C) Human platelets were activated with thrombin (0.1 U/mL) for 15 or 60 minutes, and the MPs released were isolated by ultracentrifugation. (A) Representative flow cytometry analyses of the MP population derived from platelets activated with thrombin for 60 minutes. Because of the size of heterogeneity of the MPs,⁴⁹  fluorescent Sky Blue microspheres, ranging from 90 nm to 3200 nm in diameter (Spherotech, Lake Forest, IL), were used to calibrate our flow cytometer and estimate the size of the MPs. The CD41a+ events were portrayed as forward-scattered light (FSC) and side-scattered light (SSC) PMT graph using the BD FACSDiva software. (B) MPs were counted by flow cytometry by an FSC coupled to a PMT. Results are expressed as the mean (± standard error of the mean [SEM]) fold changes vs unstimulated platelets, used as a reference (n = 3 experiments). (C) The MP and supernatant fractions of thrombin-activated platelets were isolated and analyzed for their content in miR-223 by qPCR. Results were normalized by the 2^{^-ΔΔCt} method, using RNU6 as a reference,²⁷  and expressed as the mean (±SEM) fold changes vs unstimulated platelets (n = 3 experiments). Similar results were obtained by monitoring 4 additional microRNAs (data not shown). *P < .05 vs baseline (Student t test). SSC, side-scattered light.