Anti cd16 32 mab

Tumors were excised, cut with scissors, and were subsequently incubated with a solution composed of 1 mg/mL collagenase IV (Sigma) and 0.02 mg/mL DNAse I (Sigma) in serum-free RPMI medium for 45 min at 37°C on a heating rotating plate (190 rpm). Cell suspensions were filtered using a 40 μm cell strainer and washed with 20 mL cold FACS buffer (PBS 2% FBS, 5mM EDTA). Cells were centrifuged at 300g for 5 min, and resuspend in cold FACS Buffer, filter through 5 mL round-bottom polystyrene test tubes with cell strainer cap (ThermoFisher) and kept on ice until transfer into 96 U-bottom plates (ThermoFisher) for staining. Following Fc block (anti-CD16/32 mAb (BD Bioscience) for 10 minutes at 4°C), antibodies against cell surface antigens were added (30 minutes incubation at 4°C). For staining of intracellular antigens cells were fixed, permeabilized (eBioscience FoxP3/Transcription Factor Staining Buffer Set) and stained with antibodies following manufacturer’s provided protocol (Supplementary Table S1, “FACS antibodies”). FMO controls were always included as negative controls. To detect OVA-specific CD8⁺ T cells, we used OVA257–264 (SIINFEKL) peptide bound to H-2Kb monoclonal antibody (eBio25-D1.16 (25-D1.16))-APC (from eBioscience, cat #17–5743-82). Flow cytometry was performed on BD LSR Fortessa (BD Bioscience), and FlowJo software was used for analysis.

Mice were sacrificed and bone marrow extracts, spleens and skins were collected. Spleens were injected with collagenase D (Sigma) in media (αMEM + 5% FBS + P/S). Total skin was minced using scissors and incubated with digestion buffer (media + 1 U/ml dispase) for 2 hrs at 37°C, followed by addition of collagenase D for 30 min. Digestion was stopped by adding 10 mM EDTA. All tissues were passed through 70-µm cell strainers (BD). Cells were treated with ammonium chloride buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 100 µM EDTA) to lyse erythrocytes. Cells were pretreated with anti-CD16/32 mAb for 10 min (to block non-specific binding (BD Biosciences). The cells were stained with anti-CD45 (30-F11, APC), anti-CD11b (M1/70, PB), Annexin V (APC), 7-AAD, anti-Gr-1 (RB6-8C5, PeCy7), anti-Ly6G (1A8, FITC, APC or Pacific Blue; 1A8), anti-CXCR2 (TG11/CXCR2, Alexa 647), and isotype controls were all obtained from Biolegend. Anti-IL-17RA (PAJ-17R, PE) was purchased from ebioscience. AccuCheck counting beads (Life Technologies) were used to determine absolute cell number per cm² based on the manufacturer’s protocol. Cells were analyzed on a BD FACS ARIA flow cytometer (BD Biosciences). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).