Wkymvm peptide

NADPH oxidase activity was measured by luminol/isoluminol-enhanced chemiluminescence (CL) using a microplate reader (Synergy H1 MultiMode Reader, BioTek, U.S.A) [30] . For intracellular ROS measurement, granulocytes were added to KRG-Ca 2+ containing cell permeable luminol (10 µg/mL; Sigma-Aldrich) and 50 U/mL superoxide dismutase (Solarbio) to scavenge extracellular superoxide, while extracellular ROS was measured in the presence of 10 µg/mL isoluminol (Sigma-Aldrich). Neutrophils and reagents were then incubated for 5 min at 37°C before stimulation with fMLP (1 µM; Sigma-Aldrich), WKYMVM peptide (1 µM; Sigma-Aldrich), or phorbol 12-myristate 13-acetate (PMA) (500 nM; Sigma-Aldrich). CL was recorded continuously for 20-25 min.

Peripheral blood neutrophils were isolated as recorded previously [27] . After isolation, granulocytes were kept on ice in KRG buffer (pH 7.3; 120 mM NaCl, 5 mM KCl, 1.7 mM KH 2 PO 4 , 8.3 mM NaH 2 PO 4 , 10 mM glucose), supplemented with Ca 2+ (1 mM) and Mg 2+ (1.5 mM). For assessment of neutrophil activation and degranulation, cells were left untreated at room temperature or incubated for 20 min at 37℃ with or without N-Formylmethionyl-leucyl-phenylalanine (fMLP) (50 nM; Sigma-Aldrich), tumor necrosis factor α (10 ng/mL; Sigma-Aldrich), or WKYMVM peptide (50 nM; Sigma-Aldrich). Granulocytes were xed and erythrocytes lysed by treatment with FACS lysing solution (BD Biosciences) for 15 min at 4℃. Cells were then stained with anti-CD11b, anti-CD62L, anti-CD35, anti-CD63, or anti-CD66b antibodies (all from BD Biosciences) for 1 h on ice. A minimum of 10,000 neutrophils were assessed by ow cytometry [28] .