Cd16 32 antibody

Antibodies used in this study for FACS analysis and sorting are listed in Supplementary Table 1. Red blood cells were lysed with ammonium chloride. CD16/32 antibody (BD Biosciences) was used to block the non-specific binding to Fc receptors before all stainings. Dead cells were excluded using DAPI (Invitrogen). For Tconv and Treg isolation from Foxp3^EGFP reporter mice, CD45⁺TCRβ⁺CD4⁺ T cells, respectively GFP⁻ and GFP⁺, were FACS-sorted (BD FACS ARIA^TM IIu). Nrp1 staining was added to verify that the proportion of Nrp1^−/low “adaptive” pTregs was substantially increased in the intestines compared to the spleen. For ILC3 isolation, CD45^+/low lineage-negative (CD11b⁻CD11c⁻CD19⁻TCRαβ⁻TCRγδ⁻) RFP⁺NK1.1⁻ cells were FACS-sorted from the intestines of RORγt-fate map mice (Rorc(γt)-Cre^TG × Rosa26-tdRFP). Lineage-negative NK1.1⁺ ILC1s were FACS-sorted from spleen or intestines. CD11b⁺CD11c⁺ myeloid cells were FACS-sorted from the spleen. TCRγδ⁺ and TCRβ⁺CD4⁺ T cells were FACS-sorted from inguinal lymph nodes of mice developing psoriasis-like dermatitis. All populations were isolated (>10⁵ cells per mouse) to a purity of >98%.

The brain slices were prepared as previously published [17 (link)]. After being blocked with 5% goat serum (TBST), the brain slices were incubated with CD16/32 antibody (1:500, BD Pharmingen, San Jose, CA, USA), CD206 (1:500, Abcam, Cambridge, UK), and Iba-1 (1:500, Wako, Richmond, VA) overnight at 4°C to 8°C. The sections were then treated with a fluroscencesecondary antibodies (1: 500, Invitrogen), and images were taken using a fluorescencemicroscope (Olympus PX51, Olympus Corporation, Shinjuku-ku, Japan) and analyzed with Adobe Photoshop 5.5 software. The cell number calculation was determined by counting of three randomly selected microscopic fields across three slides in the penumbra of ipsilateral cortex. Data are expressed as mean ± SD number of the percentage of CD16/32+ or CD206+ cells to the Iba-1+ cells.

Immune cells were detected via CyTOF according to the manufacturer's manual. In brief, cells in a single cell suspension from tumour tissues were counted, and 3 × 10⁶ were first incubated with cisplatin (Fluidigm Sciences). For staining, cells were blocked with 50 µl CD16/32 antibody (BD Biosciences) for 15 minutes at room temperature, and then, 50 µl CyFACS buffer containing a surface antibody cocktail was added directly to the blocking solution and incubated with cells for 30 minutes at room temperature. The metal-labelled antibodies are listed in Table S1. Next, the cells were successively fixed with 1.6% paraformaldehyde (Fluidigm Sciences) and incubated with intercalator-Ir (Fluidigm Sciences). After incubation overnight at 4 ℃, the cells were washed twice and resuspended in 1 × EQ Four Element Calibration Beads (Fluidigm Sciences). Finally, 300,000 events were collected on a CyTOF mass cytometer (Fluidigm Sciences). The data were analysed on the Cytobank website after normalization.

4-week-old male Kunming mice were purchased from the Experimental Animal Center of Fourth Military Medical University and approved by the Animal Ethics Committee of Fourth Military Medical University (License number: IACUC-2020065). The animal experiment was complied with the ARRIVE guidelines and was performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. 60% high-fat diet were purchased from Readydietech, China. Streptozocin (STZ) was purchased from Sigma, USA. Hematoxylin-Eosin (HE) was purchased from Solarbio, China. CD31 antibody was purchased from Abcam (28364, 1:100), USA. F4/80 antibody was purchased from Abcam (6640, 1:100), USA. CD16/32 antibody was purchased from BD Biosciences (553142, 1:50), USA. Fluorescence secondary antibody was purchased from Invitrogen, USA. IL-1β and TNF-α ELISA kits were purchased from Proteintech, USA.