Live dead yellow fluorescent dye

Single-cell suspensions were stained with Live/Dead yellow fluorescent dye (1:1,000, ThermoFisher) for 15 min at room temperature and washed with FACS buffer at 300xg for 5 min at 4^oC. Blocking followed with 5 ul human FcR blocking reagent (Miltenyi Biotec) per 100 ul cell suspension for 15 min on ice and staining for blood immune cells was performed with the following anti-human antibodies for 30 min on ice: CD3 (1:54; UCHT1; Biolegend), CD3 (1:27; UCHT1; BD Biosciences), CD4 (1:54; RPA-T4; Biolegend), CD8 (1:54; SK1; Biolegend), CD11c (1:32; 3.9; Biolegend), CD14 (1:68; HCD14; Biolegend), CD14 (1:32; M5E2; BD Biosciences), CD16 (1:68; 3G8; Biolegend), CD19 (1:45; HIB19; BD Biosciences), CD19 (1:32; HIB19; Biolegend), CD33 (1:54; HIM3-4; BD Biosciences), CD45 (1:45; HI30; Biolegend), CD56 (1:27; B159; BD Biosciences), CD56 (1:32; NCAM16.2; BD Biosciences), CD66b (1:54; G10F5; Biolegend), CD123 (1:32; 6H6; Biolegend), CD127 (1:32; HIL-7R-M21; BD Biosciences), HLA-DR (1:68; L243; Biolegend) and Siglec-8 (1:21; 7C9; Biolegend). Cells were centrifuged at 300xg for 5 min at 4^oC and resuspended in 1 ml FACS buffer. Data acquisition was performed on a 3 laser-FACS Aria III cell sorter (BD Biosciences) and were analyzed with FlowJo v10 software (BD Biosciences). Sorted neutrophils (40,000) were frozen at -80^oC for further processing.

Single-cell suspensions were stained with Live/Dead yellow fluorescent dye (Thermo Fisher Scientific, USA) for 15 min at room temperature and were washed with PBS at 300×g for 5 min at 4°C. They were then re-suspended in 100 µL PBS and blocked with 5 µL human FcR blocking reagent (Miltenyi, Germany) for 15 min on ice and were subsequently stained with the listed anti-human antibodies (table E4) in buffer containing PBS, 2% FCS, 1 mM EDTA for 30 min on ice. Cells were spun at 300×g for 5 min at 4 °C and re-suspended in buffer containing PBS, 2% FCS, 1 mM EDTA for analysis. Data acquisition was performed on a FACS Aria III cell sorter. Analysis was performed with FlowJo v.10 software (Tree Star, USA).