Fitc anti mouse cd11c antibody

For in vitro uptake assays, BMDCs were incubated with PBS, 5 μg/mL OVA-FITC, and 2.5, 5, and 10 μg/mL nsGO/PCP/OVA-FITC. Cells incubated with nsGO/PCP/OVA-FITC (5 μg/mL) at 4°C were used as controls. After 30 min of incubation, the BMDC cells were collected and stained with APC anti-mouse CD11c (BioLegend, CA, United States) antibody before FACS analysis. For uptake competition assays, cells were pre-treated with PCP, OVA, or 200 μg/mL mannans (Solarbio, China) for 30 min. BMDCs were then treated with nsGO/PCP/OVA-FITC (10 μg/mL) for 45 min. BMDCs were also incubated with nsGO/PCP/OVA (10 μg/mL) for 30 min, followed by co-culture with 100 μg/mL Lucifer yellow VS. dilithium salt (LY, Sigma, MO, United StatesA) for 45 min before FACS analysis. To monitor the in vivo uptake of nanoparticles, mice were injected with OVA-Alexa 647 (20 μg) or nsGO/PCP/OVA-Alexa 647 (containing 20 μg OVA) in both hind footpads. After 6 h, the popliteal lymph nodes were dissected and incubated with collagen D (Sigma, MO, United States) and DNase (Sigma-Aldrich, MO, United States) to prepare single-cell suspensions. Cells were incubated with anti-mouse CD16/32 antibody (BioLegend, CA, United States) for 15 min and then stained with FITC-anti-mouse CD11c antibody (BioLegend, CA, United States) before FACS analysis.

CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and CD3⁺CD4⁺FOXP3⁺ T cells, CD11c ⁺CD86⁺CD80⁺ dendritic cells as well as CD45⁺CD11b⁺F4/80⁺CD206⁺ M2 like macrophage and CD45⁺CD11b⁺F4/80⁺CD86⁺ M1 like macrophage in the tumor tissues or spleen were isolated and analyzed using flow cytometry. The antibodies involved in the experiment include FITC anti-mouse CD11c Antibody (Biolegend, Cat# 117306, Clone No. N418), PE anti-mouse CD86 Antibody (Biolegend, Cat# 105007, Clone No.GL-1), APC anti-mouse CD80 Antibody (Biolegend, Cat# 104714, Clone No.16-10A1), FITC anti-mouse CD3 (Biolegend, Cat# 100203, Clone No.17A2), PE anti-mouse CD4 (Biolegend, Cat# 100407, Clone No.GK1.5), APCanti-mouseCD8a (Biolegend, Cat# 100712, Clone No.53-6.7), AlexaFluor488 anti-mouse FOXP3 (Biolegend, Cat# 320012, Clone No.150D), FITC anti-mouse/human CD11b Antibody (Biolegend, Cat# 101205, Clone No. M1/70), APC anti-mouse CD45 (Biolegend, Cat# 103112, Clone No.30-F11), PerCP anti-mouse F4/80 Antibody (Biolegend, Cat# 123126, Clone No.BM8) and PE/Cyanine7 anti-mouse CD206 (Bioegend, Cat# 141720, Clone No.C068C2). The antibody concentration was 1:100 diluted with PBS.
IL-2, IL-12p70, TNF-α, and IFN-γ in primary tumors were also examined with ELISA kits. And tumors were stained for immunofluorescence of CD3⁺CD4⁺ and CD3⁺CD8⁺ proliferated CTLs in 4T1 tumor tissue slices of the primary tumor.

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.