Cd115 pe

For the analysis of platelet-leukocyte aggregates, blood was collected by retro-orbital bleeding into tubes containing 3.2% sodium citrate (9:1 v/v) (41.1506.002; Sarstedt, Nümbrecht, Germany) and kept at RT. Samples were kept at 4 °C for all subsequent steps. RBCs were lysed for 5 min (BD Pharm Lyse, BD Bioscience, Franklin Lakes, NJ, USA), samples were centrifuged, washed, and resuspended in HBSS (0.1% BSA and 0.5 mM EDTA). To assess platelet-monocyte and platelet-neutrophil aggregation, samples were incubated with a cocktail of antibodies: CD45-APC-Cy7 (557659; BD Biosciences, Franklin Lakes, NJ, USA), CD115-PE (135506; Biolegend, San Diego, CA, USA), Ly6C/G-PercP-Cy5.5 (571103; BD Biosciences, Franklin Lakes, NJ, USA), CD41-FITC (133904; Biolegend, San Diego, CA, USA) for 30 min on ice in the dark. Platelet-monocyte aggregates were identified as CD45^hiCD115^hiCD41^hi, and were further separated into platelet-Ly6C^lo monocyte aggregates, defined as CD45^hiCD115^hiLy6C^loCD41^hi, and platelet-Ly6C^hi monocyte aggregates, defined as CD45^hiCD115^hiLy6C^hiCD41^hi. Platelet-neutrophil aggregates were identified as CD45^hiCD115^loLy6G^hiCD41^hi. All samples were analyzed on an LSRII (BD Biosciences, Franklin Lakes, NJ, USA), running FACSDiVa software (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).