Comprehensive Neutrophil Phenotyping and Signaling

Harvested cells were stained with antibodies including: anti-Ly6G (1:200 dilution; BioLegend, no. 127606, 127610, or 127618), anti-CD11b (1:200 dilution; BioLegend, no. 101206), anti-CD11a (1:200 dilution; BioLegend, no. 153103), anti-SIRPα (1:200 dilution; BioLegend, no. 37210), anti-CD62L (1:200 dilution; BioLegend, no. 104428), anti-PD-L1 (1:200 dilution; BioLegend, no. 124312), anti-CXCR2 (1:300 dilution; BioLegend, no. 149604), anti-ICAM1 (1:300 dilution; BioLegend, no. 116121), anti-CD29 antibodies (1:300 dilution; BioLegend, no. 102221). In some analysis mentioned in the results, cells were treated with a fixation kit (BD Biosciences), subsequently stained with anti-STAT1 (1:100 dilution; Cell Signaling, no. 80916S), anti-p-STAT1 (Tyr701) (1:100 dilution; Cell Signaling, no. 8009S), anti-p-Src (Tyr416) (1:20 dilution; ThermoFisher, no. MA5-28055), or anti-Fyn (1:50 dilution; Santa Cruz, no. sc-434 FITC). Surface phenotype, transcription factor and intracellular protein levels of Ly6G⁺ neutrophils were analyzed using FACSCanto II (BD Biosciences). Neutrophil viability was assessed by staining and flow analysis with the annexin V/PI kit (1:4,000 dilution; Thermo Fisher Scientific, no. P3566) as described previously^{74 (link)}.