Anti mouse or anti human cd16 32 antibody

Neutrophils were washed twice in PBS after in vitro stimulation. Fc receptors were blocked for 20 min at RT with anti-mouse or anti-human CD16/32 antibody (eBiosciences 16-0161-81) followed by incubation with FITC tagged anti-Ly6G (clone 1A8, Biolegend, 127613, 0.5 μg added to 1 × 10⁶ murine neutrophils in 100 μl) and anti-P2X₇ receptor antibodies as described above. F(ab')2 donkey anti-rabbit IgG-PE (eBiosciences) was used as secondary antibody for P2X₇. Following two washes in FACS Buffer (1% FBS in PBS), cells were fixed in 0.5% PFA for analysis by flow cytometry using an Accuri C6 Flow cytometer (Becton Dickinson).

Flow cytometry was performed to determine the proportion of neutrophils in peripheral blood at different stages of fungal keratitis. Cell blood samples were washed twice at 1000 rpm for 5 min at 4°C in PBS. The cell suspension was resuspended in Thermo lysing buffer for 60s to eliminate red blood cells out of cell suspension, and it was centrifuged again at 1000 rpm for 5 min at 4°C in PBS and then stained with antibodies in the dark. Fc receptors were blocked for 15 min at RT with anti-mouse or anti-human CD16/32 antibody (e-Bioscience 16-0161-81) followed by incubation with FITC tagged anti-CD11b antibody and PE tagged anti-CD66b antibody (Bio-legend, 127613, Bio-legend, 561650, 0.5 μg added to 1 × 10⁶ cell suspension in 100 μL). After two washes in FACS buffer (1% FBS in PBS), the cells were fixed in 0.5% PFA, then washed, resuspended in flow cytometry staining buffer and analyzed by flow cytometry on a FACs Aria (BD Biosciences). Data were analyzed using BD FACSDiva software.