Identification of TF positive platelets by flow cytometry and confocal microscopy. Platelet rich plasma from a healthy volunteer was activated with TRAP-6 for 15 minutes at room temperature in the presence of CD41-PerCPcy5.5 (BD Biosciences), CD62P-PE (P-selectin, BD Biosciences) and TF-FITC (American Diagnostica) mAbs, analyzed and sorted by BD FACSAria IIU (561-488-633-355 laser equipped) and BD FACSDiva software. The following gating strategy for the identification and sorting of TF⁺ platelet was used (population hierarchy in panel I). Platelets were identified according to their physical parameters, FSC and SSC (panel A) and to the expression of the platelet marker, CD41 (panel B). The quadrant gate in panel C defines the CD62P/TF negative population in resting platelets. This marker has been copied on subsequent dot plots. Panel D shows TF/CD62P positivity on activated CD41⁺ cells. Cross excitation risk was avoided by using 488 and 561 lasers for FITC and PE, respectively. TF⁺ platelet aggregates were further removed through doublets exclusion in FSC/TF and SSC/TF dot plots (panels E and F) and double positive TF/CD62P single events are shown in panel G. Highly fluorescent events (P3, panel H) were sorted and analyzed by confocal microscopy (panel J; LSM 710 Zeiss, objective Plan-Apochromat 100×/1.4 oil).