Apc conjugated anti mouse ly6g antibody

To deplete neutrophil, mice were treated with intraperitoneal (i.p.) injection of 150 µl (1 mg/kg) anti-Gr-1 antibody (Clone RB6-8C5, BE0075, BioXcell, NH) to deplete neutrophils 24 hours prior to the CLP surgery 38 (link). To verify neutrophil depletion, blood neutrophil levels were assessed by flow cytometry operated on FACSCanto II flow cytometer (BD Bioscience, San Jose, CA, USA) with APC-conjugated anti-mouse Ly6G antibody (127613, BioLegend, San Diego, California, USA) and FITC-conjugated anti-mouse CD11b (101205, BioLegend, San Diego, California, USA). The data were analyzed with FlowJo V7.6 software (Tree Star, Inc., Ashland, OR).

Cells were blocked with purified rat anti–mouse CD16/CD32 Fc block (BD Biosciences, catalog No.553142) at 4°C for 15 minutes, prior to incubation with various fluorescence-conjugated antibodies at 4°C for 30 minutes. Cells were washed twice after antibody incubation. A single-stained control for each fluorochrome was made for compensation for each experiment. Labeled cells were detected by flow cytometry with FACS Calibur (BD Bioscience) or FACS Canto II instruments (BD Biosciences) and analyzed with FlowJo software (TreeStar). Mouse Gr-1 FITC conjugated antibody (Invitrogen, REF. No. RM3001), APC conjugated anti-mouse Ly6G antibody (BioLegend, catalog No.127613), PerCP/Cyanine5.5 conjugated anti-mouse CD274 antibody (PD-L1; BioLegend, catalog No.124333) were used.

The peritoneal cavity of each mouse was washed twice with 5 mL of PBS to fully extract the peritoneal exudate, and a single cell suspension was still isolated with a cell filter. Single‐cell suspensions were isolated from the spleens and the peritoneal exudates of mice from the control group and ATG groups by grinding and filtering the tissues through a cell strainer. Some of these cells were stimulated for 4 hours with Cell Stimulation Cocktail (plus protein transport inhibitors) (500×) (eBioscience) in an incubator at 37℃ with 5% CO₂. Then, the cells were fixed, permeabilized, and stained for intracellular cytokines with a PE‐conjugated anti‐mouse IL‐6 (Sungene Biotech, Tianjin; Cat# M100I5‐09C; clone MP5‐20F3) or PE‐conjugated anti‐mouse TNF‐α antibody (Sungene Biotech; Tianjin; Cat# M100T12‐09D; clone XT311). Other cell surface markers were assessed with a FITC anti‐mouse CD11b antibody (Sungene Biotech; Cat# M10117‐02E; clone M1/70), a PE‐conjugated anti‐mouse CD62L antibody (BD Biosciences; Cat# 553151; clone MEL‐14), a APC‐conjugated anti‐mouse F4/80 antibody (Sungene Biotech; Cat# M100F1‐11C; clone BM8) and an APC‐conjugated anti‐mouse Ly6G antibody (BioLegend; Cat# 108411; clone RB6‐8C5). Nonspecific expression was detected with a homotypic control. The average fluorescence intensity (MFI) was analyzed using FlowJo 7.6 software (BD Biosciences).