Apc anti mouse cd86 antibody

For the characterization of differentiated macrophages, APC anti-mouse CD86 Antibody (dilution 1:100, BioLegend, 105012), anti-mouse CD206 antibody (1:100, BIO-RAD, MCA2235) conjugated with Goat anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:500, Invitrogen, A-11006), Human CD38 Alexa Fluor® 488-conjugated Antibody (1:100, R&D Systems, FAB2404G), and CD36 Monoclonal Antibody (1:100, Invitrogen, MA5-14112) conjugated with Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (1:500, Invitrogen, A32723) were incubated with the cells for 30 min, and Hoechst33342 (1 μg/mL) was added for 5 min prior to the experiment. The images were taken by Operetta High Throughput Screening (Perkin Elmer, Walthan, MA, USA), and the data were collected from Harmony 4.8 (Perkin Elmer).

Orthotopic tumor-bearing mice (3 per group) were taken at the end of the above treatment and euthanised. Mouse lymph nodes and tumor tissue were taken, ground, and placed in staining buffer (Multisciences (Lianke) Biotech, Co., Ltd. Lot. A10752) through a 70 μm pore size filter (Thermo Fisher Scientific). The above cell suspension was centrifuged (200 × g, 5 min) and the supernatant was removed. Staining buffer was added to blow the cells well. First 10 μL of FC blocking (1 μg/10⁶ cells, Miltenyibiotec, Lot. 5210508639) was added to each sample tube, and they were shaken and mixed well to avoid non-specific antibody binding. After incubation on ice for 30 min, the supernatant was discarded by centrifugation (200 g, 5 min), and 100 μL of staining buffer was added for staining. Mature DCs in lymph nodes were detected by staining on ice for 45 min using FITC-anti-mouse CD11c antibody (dilution 1:200, catalog number: 117305, clone: B277031, Lot: N418, Biolegend), PE-anti-mouse CD80 antibody (dilution of 1:40, catalog number: 104707, clone: 16-10A1, Lot: B340153, Biolegend) and APC-anti-mouse CD86 antibody (dilution of 1:80, catalog number: 105071, clone: GL-1, Lot: B323580, Biolegend). The cells were resuspended with 100 μL of staining buffer, filtered through non-woven fabric, and placed in a flow cytometer for analysis.

BMDMs with different phenotypes were cultured for repolarization assay. M2-like macrophages were incubated with PBS, IMDQ, NPGN, PGN_4.9 nanoadjuvants (equivalent to 10 μM IMDQ) and PGN_4.9 control polymer (w/o IMDQ) in DMEM medium for 24 h. For cell morphology study, BMDMs were stained with AF488-wheat germ agglutinin (WGA, Invitrogen), Hoechst 33342 and TRITC Phalloidin (YEASEN) for cell membrane, nucleus and cytoskeleton labelling, respectively. The fluorescence images were visualised by a confocal laser scanning microscope (ZEISS LSM880) under a 63× oil objective lens.
To image the expression of M1 and M2 markers, BMDMs were stained with APC anti-mouse CD86 antibody (105012, BioLegend, clone number: GL-1, Dilution 1:80), and PE anti-mouse CD206 antibody (141706, BioLegend, clone number: C068C2, Dilution 1:40) according to the manufacturer’s instructions. The statistical results were evaluated by flow cytometry. The supernatant was collected for analysis of proinflammatory cytokines IL-12 using ELISA kits (Peprotech).

MC38.OVA tumour-bearing mice were injected intravenously with PBS, IMDQ, NPGN and PGN_4.9 nanoadjuvants (equivalent to 2 mg kg^-1 IMDQ) every four days for three times. Four days after the last treatment, popliteal lymph nodes and inguinal lymph nodes were dissected and prepared into a single-cell suspension. The cells were stained with Brilliant Violet 510 anti-mouse F4/80 (123135, Biolegend, clone number: BM8, Dilution 1:40), PE-Cy7 anti-mouse CD80 antibody (104734, Biolegend, clone number: 16-10A1, Dilution 1:40), APC anti-mouse CD86 antibody (105012, Biolegend, clone number: GL-1, Dilution 1:80), and PE anti-mouse SIINFEKL-H-2K^b antibody (116608, Biolegend, clone number: SF1-1.1, Dilution 1:40) and detected by flow cytometry. Meanwhile, tumour tissues were harvested and digested by a tumour dissociation kit (Miltenyi Biotec). The single-cell suspension in different groups was stained with PerCP/Cy5.5 anti-mouse CD45 (103132, Biolegend, clone number: 30-F11, Dilution 1:100), FITC anti-mouse/human CD11b (101205, Biolegend, clone number: M1/70, Dilution 1:200), Brilliant Violet 510 anti-mouse F4/80 (123135, Biolegend, clone number: BM8, Dilution 1:40), and PE anti-mouse SIINFEKL-H-2K^b antibody (116608, Biolegend, clone number: SF1-1.1, Dilution 1:40). SIINFEKL⁺ cells in CD11b⁺F4/80⁺ macrophages were measured by flow cytometry.