Clone g10f5

Peripheral blood was collected in EDTA vial, and 200 μl of blood was aliquoted for antibody staining. Briefly, 1 ml of RBC lysis buffer was added to 200 μl of whole blood, and mixed properly. A 10× red blood cell lysis buffer was prepared in-house using NH₄Cl (0.155 M), KHCO₃ (0.01 M) and EDTA (0.1 mM). The tubes were incubated for 10 min at 4 °C, and centrifuged at 300×g for 10 min. The cell pellets were washed twice with 1× PBS and suspended in 300 μl of PBS to obtain a single cell suspension. For cell surface staining, 100 μl of cell suspension was used, and the antibodies CD16 (1:100) (clone 3G8, APC conjugated, Cat No 302011, Biolegend, USA), CD66b (1:100) (clone G10F5, FITC conjugated, Cat No 305103, Biolegend, USA) and CD177 (1.5:100) (clone MEM-166, APC/Cyanine 7 conjugated, Cat No 315809, Biolegend, USA) was used. Unstained controls, stained samples, and fluorescence minus one controls were acquired on BD-LSR Fortessa flow cytometry machine. Using the FACS DIVA software, PMN gating was done based on FSC-A v/s SSC-A plot. Enriched and activated neutrophils were gated based on CD16⁺ CD66b⁺ (double positive) in a quadrant plot. CD177⁺ neutrophils were gated within these double positive cells.

Isolated neutrophils (50,000 cells) were stained in fluorescent activated cell sorting (FACS) buffer containing 2% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Dun Laoghaire, Ireland) and 1 mM EDTA (Life Technologies) in phosphate buffer saline (PBS) without calcium and magnesium (Corning, Corning, NY, USA). Cells were stained with antibodies for 30 min at 4 °C in FACS buffer containing (BV421) anti-CD10 (1:200 dilution; clone HI10a; BioLegend, San Diego, CA, USA) and (BV786) anti-CXCR4 (1:100 dilution; clone12G5; BioLegend) and (AF647) anti-CD64 (1:100 dilution; clone 10.1; BioLegend) or (BV786) anti-CD63 (1:100 dilution; clone H5C6; BioLegend) and (APC) anti-CD66b (1:800 dilution; clone G10F5; BioLegend), or (BV421) anti-CD62L (1:200 dilution; clone DREG-56; BioLegend), and (PE/Dazzle) anti-CD32 (1:200 dilution; clone FUN-2; BioLegend). Data were acquired on a BD FACSCelesta (BD Biosciences, San Jose, CA, USA) with a BVR laser configuration (488 nm, 405 nm, 640 nm). Before recording data, gates were prepared so that 10,000 neutrophil events could be collected. FCS files were exported from BD FACSDiva Software (BD Biosciences) in a 3.0 format. FCS files were analyzed using FlowJo v.10 software (BD Biosciences).