Pe cy7 anti mouse cd86

Carprofen (Rimadyl^® Injectable, 50 mg/mL) was purchased from Patterson Companies (Saint Paul, MN, USA). Pacific Blue™ anti-mouse CD3, FITC anti-mouse CD4, PerCP anti-mouse CD8a, PE anti-mouse/human CD11b, APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1), PE/Cy7 anti-mouse CD62L, APC anti-mouse/human CD44, APC anti-mouse CD25, Biolegend PE anti-mouse/rat/human FOXP3, PerCP anti-mouse CD11c, PE/Cy7 anti-mouse CD86, FITC anti-mouse I-A/I-E, and APC anti-mouse CD40 were purchased from Biolegend (San Diego, CA, USA). Mouse tumor dissociation kit (No. 130-096-730) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

To test whether the Ncom Gel vaccine could promote the differentiation of M0 macrophages to M1 macrophages in vitro, RAW264.7 cells were seeded in 24 plates (Nest Biotech, Wuxi, China). Then, free OVA, OVA plus CpG solution (OVA/CpG), OVA plus NOCC-CpG (OVA/NOCC-CpG), OVA plus OX-M (OVA/OX-M) and OVA encapsulated hydrogel (OVA/Ncom Gel) were added and the cells were cultured for 24 h. The formulation concentrations were equal to those used for the BMDC experiments. Next, the cells were obtained and stained with FITC anti-mouse CD80, PE-Cy7 anti-mouse CD86 and APC anti-mouse CD40 (Biolegend, San Diego, CA, USA). PE anti-mouse F4/80 was used to stain every aliquot of cells as a marker of macrophages. Finally, these cells were analyzed by flow cytometry (ACEA NovoCyte, San Diego, California, USA). To evaluate whether the Ncom Gel vaccine could reduce M2 macrophages in vitro, RAW264.7 cells were cultured with IL-4 for 24 h to induce M0 macrophages to differentiate into M2 macrophages. Next, free OVA, OVA plus CpG (OVA/CpG) and OVA encapsulated hydrogel (OVA/Ncom Gel) were added to cells for another 24 h incubation. Finally, the obtained cells were stained with PE anti-mouse F4/80 and APC anti-mouse CD206 (Biolegend, San Diego, CA, USA) and analyzed by flow cytometry (ACEA NovoCyte, San Diego, California, USA).

Antibodies for immunofluorescence were purchased from the following sources: F4/80 (0.2 μg/ml, BM8, OriGene, Rockville, MD, USA), TNF-α (2.5 μg/ml, MP6-XT3, eBioscience, San Diego, CA, USA), IL-10 (2.5 μg/ml, JES5-2A5, eBioscience), fibronectin (0.7 μg/ml, Polyclonal, invitrogen, Carlsbad, CA, USA), and collagen IV (2.5 μg/ml, Polyclonal, Invitrogen). Secondary antibody were goat anti-rat IgG Alexa Fluor® 488 (2 μg/ml, BioLegend, San Diego, CA, USA), streptavidin Alexa Fluor® 594 (0.5 μg/ml, BioLegend), goat anti-rabbit IgG Alexa Fluor® 488 (2 μg/ml, Invitrogen).
The following antibodies (BioLegend) were used for flow cytometry: APC anti-mouse F4/80 (2.5 μg/ml, BM8), PE/Cy7 anti-mouse CD86 (1.25 μg/ml, GL-1), PE anti-mouse CD206 (1.25 μg/ml, C068C2), PE/Cy7 anti-mouse TNF-α (5 μg/ml, MP6-XT2), and Alexa Fluor® 488 anti-mouse IL-10 (12.5 μg/ml, JES5-16E3).