Population analysis of multicolor flow cytometry data with platelet surface and activation markers on the size-separated platelet fractions. Isolated, size-separated platelets were stimulated with 5 µg/mL CRP-XL, 20 µM TRAP-6, or 5 µM 2-MeSADP and stained simultaneously for PAC1, GPVI, CD36, CD63, and GPIX for 15 minutes before fixation and measuring 10 000 events per sample. Clustering tools (FlowSOM) and a dimensionality reduction algorithm (tSNE) were used to analyze and visualize these multicolor flow cytometry data. (A) Representative tSNE plots with a FlowSOM population filter of the different platelet fractions (unsorted, large, and small) under different stimulations. A heatmap showing the individual profiles of platelet populations per sample is shown in supplemental Figure 2. (B) Characterization of the FlowSOM clusters (5 populations; 1-5). (C-E) Distribution profile of platelet populations upon stimulation with CRP-XL (C), TRAP-6 (D), or 2-MeSADP (E), respectively, resulting from FlowSOM analysis for the different platelet size fractions. Mean ± standard error of mean; n = 4. *P < .05.