Cd41a fitc

All procedures and protocols were approved by the Scientific Research Ethics Committee of the Fourth Military Medical University. All experiments were performed in accordance with the principles and guidelines of the Care and Use of Laboratory Animals. Wildtype C57BL/6 mice (WT) were obtained from the experimental animal center of Fourth Military Medical University. CD226^fl/fl mice with C57BL/6 background were constructed by the Cyagen company (Suzhou, China). Platelet factor 4 (PF4)-Cre mice were obtained from the model animal research center of Nanjing University. CD226^fl/fl mice were breed with PF4-Cre mice to acquire CD226^fl/flPF4-Cre mice with specific knockout of CD226 in megakaryocytes (MKs)/platelets. The CD226^fl/fPF4-Cre mice were genotyped via classical polymerase chain reaction (PCR) for PF4-Cre (forward primer sequence: CCC​ATA​CAG​CAC​ACC​TTT​TG; reverse primer sequence: TGCACAGTCAGCAGGTT) in DNAs extracted from tail, and CD226 deletion was checked via flow cytometric analysis in CD41⁺ platelets isolated from mice (antibodies used were CD41a-FITC and CD226-APC (eBioscience, CA, United States). The mice were housed under specific pathogen-free conditions in standard, with individually ventilated cages, and were fed with standard laboratory chow and water. All mice that were used in this study were 8–12 weeks old (weight 23–26 g).

Aliquots of blood plasma 16,000g and 160,000g pellets (fractions III and IV) were stained with mouse anti-human CD3-FITC, CD79a-PerCP-Cy5.5, CD41a-FITC, CD34-APC, and CD63-PE according to the manufacturer's recommendations (eBioscience, USA). Pellets were washed twice with dfPBS and analysed by FACSCanto II (Becton Dickinson, USA). Forward scatter and side scatter (FSC and SSC) PMT voltage settings were adjusted for the detection of blood vesicles/extracellular particles (60–1000 nm) using CST beads (Becton Dickinson, USA) and 60 nm polystyrene beads (Thermo Scientific, USA). PMT voltage settings for the detection of FITC, PE, PerCP-Cy5.5, and APC fluorescence were adjusted using Anti-Mouse Ig, κ/Negative Control Compensation Particles Set beads, according to manufacturer's recommendations (Becton Dickinson, USA). The following settings were used for flow cytometry analysis: FSC, 615; SSC, 310; FITC, 548; PE, 466; PerCP-Cy5.5, 505; APC, 300; threshold FSC/SSC, 200/200; compensation PE-FITC, 18; and compensation PerCP-Cy5.5/PE, 15. Gates were set according to unstained samples. Flow cytometry data were analysed with BD FACSDiva software v6.1.3 (Becton Dickinson, USA). Overlaid histograms were created using Flowing software v2.5.1 (Turku University, Finland).

Preparations of EVs obtained at 160,000 g were stained with mouse anti-human CD3-FITC, CD79a-PerCP Cy5.5, CD41a-FITC, CD34-APC, and CD63-PE according to the manufacturer’s protocol (eBioscience, San Diego, CA, USA). Pellets were twice washed with dfPBS and analyzed using FACS Canto II (Becton Dickinson, Franklin Lakes, NJ, USA). Forward scatter and side scatter (FSC and SSC) PMT voltage settings were adjusted for the detection of extracellular particles (60–1000 nm) using CST beads (Becton Dickinson, USA) and 60 nm polystyrene beads (Thermo Scientific, Waltham, MA, USA). PMT voltage settings for the detection of FITC, PE, PerCP-Cy5.5, and APC fluorescence were adjusted using “Anti-Mouse Ig, κ/Negative Control Compensation Particles Set beads”, according to the manufacturer’s protocol (Becton Dickinson, Franklin Lakes, NJ, USA). The following settings were used for the flow cytometry analysis: FSC, 615; SSC, 310; FITC, 548; PE, 466; PerCP-Cy5.5, 505; APC, 300; threshold FSC/SSC, 200/200; compensation PE-FITC, 18; and compensation PerCP-Cy5.5/PE, 15. Gates were set according to unstained samples. Flow cytometry data were analyzed with BD FACSDiva software v6.1.3 (Becton Dickinson, USA). Overlaid histograms were created using Flowing software v2.5.1 (Turku University, Turku, Finland).