Platelet Activation and Surface Marker Analysis

A total of 400 × 10⁶ platelets, purified as described in Section 4.1, were suspended in 1 mL of Tyrode’s buffer and stimulated with 10 or 20 µM of LL-37 (Tocris Bioscience, Bristol, UK) for 20 min at 37 °C in a 5% CO₂ atmosphere (Incubator NUAIRE, model NU-5700, Plymouth, MN, USA). After platelet simulation, the cells were obtained by centrifugation at 480 g for 10 min and the supernatant was stored at −70 °C. Subsequently, platelets were stained using mouse mAb directed against the following human molecules: anti-CD41-PE-Cy5 (Section 4.1), anti-HLA-ABC-APC (clone: G46-2.6), anti-CD86-FITC (2331 (FUN-1)), anti-CD282-APC (clone: 11G7), anti-CD32-APC (clone: FLI8. 26), anti-CD35-FITC (clone: E11) (BD Biosciences, USA), anti-PAC-1-FITC (clone: PAC-1), an-ti-CD62P-FITC (clone: AK4), anti-LOX-1-PE (clone: 15C4), anti-CD80-FITC (clone: 2D10) (Biolegend, San Diego, CA, USA), anti-CD284-APC (clone: HTA125) (Invitrogen, Waltham, MA, USA), anti-PAR1-PE (clone: 731115) (R & D System, Minneapolis, MN, USA), and anti-Dectin1-PE (clone: REA515) (Miltenyi Biotec, San Francisco, CA, USA), and using their respective mouse isotype mAbs: PE-Cy5 IgG1 κ Isotype (Section 4.1), APC IgG1 κ (clone: MOPC-21), APC IgG2b κ (clone: 27-35), FITC IgG1 κ Isotype (clone: MOPC-21), PE IgG1 κ (clone: X40), FITC IgM, κ (clone: MM-30) (Biolegend, San Diego, CA, USA), and PE IgG2b (clone: 133303) (R and D System, USA).