Neutrophil NETosis Induction by Platelet Supernatants

According to previously reported methods (69 (link)), neutrophils were purified from mouse blood samples using Ficoll-Paque (Ficoll-Paque Plus, 1.077 g/mL, GE Healthcare, Chicago, IL, USA) and dextran (Sigma-Aldrich) sedimentation (3% w/v) density gradient centrifugation and red blood cell lysis. A flow cytometer (Gallios, Beckman Coulter, Brea, CA, USA) and a fluorescent anti-citrullinated histone H3 (CitH3) antibody (Abcam, Cambridge, UK) were used to investigate the neutrophil expression of NETosis marker CitH3 after treatments of supernatants from platelets or platelets plus NDs. To prepare the platelet supernatants, inhibitors (Z-WEHD-FMK, 10 μM, R&D Systems; Z-DEVD-FMK, 10 μM, R&D Systems; OLT1177, 10 μM, Cayman Chemical; NAC 150 ng/mL, Sigma-Aldrich; MitoTEMPO, 1 μM, Sigma-Aldrich; P-selectin: rP-sel, 100 ng/mL R&D Systems; 30 min pretreatments before addition of ND) were used to block ND-induced platelet activation and cell death. After treatments with or without NDs and inhibitors, platelet supernatants were harvested by centrifugation (2.5 x 10⁴ g, 10 min; Benchtop Centrifuge, ThermoFisher Scientific) to remove platelets and NDs. Peptidyl arginine deiminase 4 (PAD4) inhibitor GSK484 (10μM, Sigma–Aldrich, St. Louis, MO, USA) was used to block neutrophil NETosis in vitro and in vivo as described (69 (link)).