Apc conjugated anti f4 80 clone bm8

Cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (5% fetal calf serum plus 5 mM EDTA in PBS) and incubated with conjugated monoclonal antibodies (mAb). The mAb used, phycoerythrin (PE)-conjugated anti–Ly-6G (clone RB6-8C5, 125931, 1/400), fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (clone M1/70, 11011241, and 1/200), and allophycocyanin (APC)-conjugated anti-F4/80 (clone BM8, 17480182, 1/800), eF450-conjugated anti-CD4 (clone RM4-5, 48004282, 1/200), PE-conjugated anti–Foxp3 (clone FJK‐16 s, 12577382, 1/100), FITC-conjugated anti-CD25 (clone PC61.5, 53025382, 1/400), APC-conjugated anti-Arginase-1 (clone A1exF5, 17369782, 1/100), APC-conjugated anti-EGR2 (clone erongr2, 17669182, 1/100), were all from eBioscience. PE-conjugated anti-CD206 (clone CD68C2, 141705, 1/100) and PE-conjugated anti-IL-6 (clone MP5-20F3, 504504, 1/100) were from Biolegend. Cells (1 × 10⁶) were incubated with appropriate conjugated antibodies for 30 min at 4 °C in the dark. Stained cells were subsequently washed twice with FACS buffer and fixed in BD CellFIX solution (BD Biosciences). Flow cytometric analyses were performed on LSRII cytometer (Becton Dickinson) using FACS Diva6 (Becton Dickinson) and FlowJoX (Tree Star) software for data processing.

Peritoneal cells were collected by lavage of the peritoneum as described above. Cells were centrifuged for 10 min at 300× g, resuspended in PBS without Ca²⁺/Mg²⁺ supplemented with 0.5% BSA (FACS staining buffer, FSB), and counted. Peritoneal cells (5 × 10⁵ cells/tube) were then incubated for 15 min with 0.5 µg of FcγR blocking antibodies (Fc block; BD Biosciences, San Jose, CA, USA), followed by antibody staining for 30 min in the dark at 4 °C. Cells were then washed, centrifuged, and resuspended in FSB for analysis. Each anti-mouse antibody was added at 0.5 µg/tube and included FITC-conjugated anti-Ly6G (clone 1A8, Biolegend, San Diego, CA, USA), PE-conjugated anti-CD11b (clone M1/70, eBioscience), PerCP-conjugated anti-CD19 (clone 1D3, BD Bioscience), APC-conjugated anti-F4/80 (clone BM8, eBioscience). Propidium iodide was also used to assess cell viability. Flow cytometry was performed using a FACSCanto II flow cytometer with FACSDiva software (BD Biosciences, San Jose, CA, USA). The data were analyzed using FlowJo software v.10.1 (Tree Star, Ashland, OR, USA).