Fitc conjugated cd11b

To detect the distribution of immune cells, we stained 1 × 10⁶ single cells with a Live/Dead Stain (1:1000; BD Biosciences, San Jose, CA, USA), Pacific blue-conjugated CD3 (1:200; BD Biosciences, San Jose, CA, USA), FITC-conjugated CD4 (1:200; BD Biosciences, San Jose, CA, USA), APC-conjugated CD8 (1:200; BD Biosciences, San Jose, CA, USA), APC-Cy™7-conjugated CD45 (1:200; Thermo Scientific, Waltham, MA, USA), FITC-conjugated CD11b (1:200; Thermo Scientific, Waltham, MA, USA), Pacific blue-conjugated MHC II (1:200; BioLegend, San Diego, CA, USA), and PE-conjugated CD86 (1:200; BD Biosciences, San Jose, CA, USA) antibodies for 30 min at 4 °C without light. The samples were processed on a BD FACSCanto II (BD Biosciences, San Jose, CA, USA), and all data were analyzed with FlowJo v10 software (Tree Star).

Cells were collected from uninured and injured tissue digest, centrifuge at 400×g for 5 min, and were resuspended in PBS with 5% bovine serum albumin. Cells were then incubated with CD16/CD32 FcBlock (BD Biosciences, 553141) for 5 min at 4 °C. Cells were labeled using a 1:100 concentration of antibodies against APC-conjugated F4/80 (Thermo Fisher Scientific, MF48005), FITC-conjugated CD11b (Thermo Fisher Scientific, RM2801), PE-conjugated Ly6G (BD Biosciences, 551461), and PerCP-Cy5.5-conjugated Ly6C (eBiosciences, 45-5932) for 20 min at room temperature in dark and then fixed with fixative medium A (Thermo Fisher Scientific, GAS004) for 15 min at room temperature. Unstained and single-stained cells controls were used as compensation controls. Cells were then washed and resuspended in 500 μL of DPBS containing 0.5% fetal bovine serum and analyzed using FACS Calibur flow cytometer (BD Biosciences). The number of gastric F480⁺/CD11b⁺/Ly6C^hi/Ly6G^neg macrophages were quantified using FlowJo software according to our previously published protocol^{14 (link)}.

To analyze the surface expression of ADAM8 on human neutrophils and PBMCs, cells were stained with a mouse monoclonal antibody against the ectodomain of ADAM8 (2.5 μg/1 million cells, R&D Systems) or respective IgG2b (R&D Systems) isotype control followed by incubation with an anti-mouse Alexa Fluor 647–conjugated antibody (5 μg/mL) as described previously (41 (link)). To determine the cell populations in the murine BAL or the peripheral blood, samples were stained using the following antibodies: eFluor450-conjugated CD11c, FITC-conjugated CD11b, PE-conjugated CD86, APC-conjugated Ly6G, PerCP-Cy5.5–conjugated CD45, and PE-Cy7–conjugated F4/80 (Thermo Fisher Scientific, BioLegend). (See complete antibody information in Supplemental Table 4.) Fluorescence intensities were recorded using Sony SH-800 flow cytometer, and data were analyzed off-line using FlowJo 10.5.3 software (Tree Star, Inc.).