Cd86 clone gl 1

After in vivo experiments, mouse tumors were rapidly excised and mechanically dissociated in PBS using scissors. The tumors were then digested in serum-free RPMI supplemented with 20 mg/ml DNase I (Roche), 20 mg/ml Dispase II (Roche) and 20 mg/ml collagenase I (Sigma) for 30–60 min at 37 °C with rotation to promote dissociation. The resulting single-cell suspensions were passed through 70 μM strainers twice, and red blood cells in the tumor samples were lysed with red blood cell lysis buffer (eBioscience, #00-4333-57) for 5 min at room temperature. Then, the single-cell suspensions were washed in Cell Staining Buffer (BioLegend) and incubated with the indicated flow antibodies at 4 °C for 30 min. Prior to staining with antibody panels, cells were blocked with a monoclonal antibody against CD16/32 (BioLegend) for 15 min at 4 °C. All the antibodies and reagents used for flow cytometry included ZombieRED (ECD, Biolegend, #423110), CD45 (clone 30-F11, Biolegend, #103116), CD3e (clone 145-2C11, Biolegend, #100328), CD4 (clone RM4-5, Biolegend, #100536), CD8 (clone 53–6.7, Biolegend, #100706), GZMB (clone QA16A02, Biolegend, #372208), PRF1 (clone S16009B, Biolegend, #154404), F4/80 (clone BM8, Biolegend, #123127), CD11b (clone M1/70, Biolegend, #101206), CD86 (clone GL-1, Biolegend, #105006) and CD206 (clone C068C2, Biolegend, #141719).

Hind limb bones (tibia and femur) were isolated shortly after the C57bl/6 male mice were euthanized. The bones were flushed with PBS using a 27 G needle and 5 mL syringe. Bone marrow was strained through a 70 µm nylon mesh filter (Fisher Scientific; Ottawa, ON, Canada) and mechanical pressure was applied using the plunger from a 5 mL syringe. Bone marrow cells were washed and red blood cells (RBC) lysed using filtered double distilled H₂O (ddH₂O) for 10 s. Cells were then counted and seeded onto a 24-well tissue culture plate at 1 × 10⁶ cells/well in RPMI media supplemented with 5% FBS and 800 U/mL of GM-CSF (Peprotech, Cranbury, NJ, USA). Plates were incubated and the medium was changed every 2–3 days according to the pH indicator of the medium. After 7–9 days of incubation, the loosely attached cells were collected, washed in PBS, and plated at 1 × 10⁶/mL/well in fresh medium with 20 × 10⁶ CFU of HKCC in 24-well tissue culture plates. Supernatant was collected 18–20 h after incubation and frozen for ELISA. Whole cells were stained for CD11c, CD86 (clone GL-1), and CD40 (clone 3/23) with fluorescently labelled antibodies (BioLegend; San Diego, CA, USA). Dendritic cell populations were >80% pure, as determined by CD11c expression.

To detect the expression levels of co-stimulatory molecules on the cell surface of RAW-267.4 cells and BMDCs, fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD40 (clone 3/23, Biolegend, San Diego, CA, USA), CD80 (clone 16-10A1, Biolegend, San Diego, CA, USA), CD86 (clone GL-1, Biolegend, San Diego, CA, USA), or MHC class II (I-A/I-E) mAbs (clone M5/114.15.2, Biolegend, San Diego, CA, USA) were used. In all stainings, IgG receptors on the cell surface were blocked using either monoclonal antibody (mAb) against CD16/CD32 (clone 2.4G2, Thermo Fisher Scientific, Waltham, MA, USA), or normal murine IgG antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). BMDCs were additionally stained with allophycocyanin (APC)-conjugated anti-mouse CD11c mAb (clone N418, Biolegend, San Diego, CA, USA) to gate the DC population. Fluorescence intensity of stained cells (10,000 cell detection) was measured with flow cytometry using a FACSLyric (BD Bioscience, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo V. 7 (BD Bioscience, Franklin Lakes, NJ, USA).