Apc anti mouse cd206

For surface staining, the cell suspension was incubated with fluorescently labeled antibody at room temperature for 20 minutes. For CD206 staining, the samples were fixed and permeabilised with BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences, US), and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. For intracellular TNF-α staining, the samples were stimulated by LPS (100 ng/ml, Beyotime) for 4 h, fixed and permeabilised, and then incubated with fluorescently labeled antibody at 4 °C for 35 minutes. Bacteria were stained using the BacLight™ Red kit (Invitrogen, B-35001). miRNA was labelled with fluorescein amidites (FAM). The monoclonal antibodies used were as follows: APC-Cy7-anti-Mouse F4/80, Pacific Blue-anti-Mouse CD11b, FITC-anti-Mouse CD11c, APC-anti-Mouse CD206, APC-Cy7-anti-Mouse TCRβ, APC-anti-Mouse NK1.1, PerCP-Cy5-5-anti-Mouse CD45, PE-Cy7-anti-Mouse CD3, PE-anti-Mouse CD4, FITC-anti-Mouse CD8, APC-anti-Mouse TNF-α, all purchased from Biolegend; FITC-anti-Human CD86 (20 μl/ test), APC-anti-Human CD206 (20 μl/test), purchased from BD Pharmingen. Flow cytometry or Image Stream was conducted using a BD FACS CantoII or Millipore ISX with fluorochrome-conjugated cells, and the data was analysed using FlowJo software version 10.4 or IDEAS version 6.0.

After stimulation for 24 hrs, the immunophenotype of RAW 264.7 cells was evaluated by flow cytometry, as described previously 31. Briefly, 1 × 10⁶ M0‐unpolarized, M1‐polarized or M2‐polarized Mφs were trypsinized and washed twice with cold PBS. The cell suspension was then divided into sterile Eppendorf tubes and blocked with 2% anti‐mouse CD16/32 (Biolegend, San Diego, CA, USA) on ice for 10 min. The cells were then washed twice and incubated with the following antibodies for half an hour in the dark: PE anti‐mouse CD86 and APC anti‐mouse CD206 (both from Biolegend). Cells that were not pretreated with any antibody were used as blank controls. The cells were washed twice to remove excess antibodies, resuspended in 400 μl PBS containing 3% FBS and analysed with a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, USA).

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).

For flow cytometry analysis, the tumor mass was dissociated into single cells. Prior to antibody staining, red blood cells were removed with ammonium chloride-potassium lysis buffer for 3 min at room temperature, followed by incubation or staining with cell surface antibody [APC anti-mouse CD206 (BioLegend)] for 30 min on ice. The cells were then washed twice and re-suspended in FACS buffer. Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter), and the resulting data were analyzed using CytExpert software.

DMEM was purchased from BasalMedia. Penicillin/streptomycin and FBS were acquired from Gibco. TRIzol reagent was obtained from Thermofisher. 5x HiScript II Q RT SuperMix and 2x AceQ Universal SYBR qPCR Master Mix were purchased from Vazyme. Recombinant murine M-CSF, IFN-γ, and IL-4 protein were purchased from Peprotech. The antibodies are listed as follows: p-STAT1 (Abclonal), STAT1 (Abclonal), p-STAT3 (Abclonal), STAT3 (Cell Signaling Technology, CST), p-STAT6 (Abcam), STAT6 (Abclonal), p-ERK (CST), ERK (CST), p-JNK (CST), JNK (CST), p-P65 (CST), P65 (CST), p-P38 (CST), P38 (Abclonal), iNOS (CST), Arg1 (CST), PRMT2 (Novus), β-actin (CST), PE anti-mouse F4/80 (Biolegend), FITC anti-mouse CD86 (Biolegend), APC anti-mouse CD206 (Biolegend).

For surface markers, the cells were stained in PBS containing 1% BSA with indicated antibodies for 30 min on ice. For intracellular markers, the cells were first fixed with Fixation Buffer (420801; Biolegend, San Diego, CA, USA) at 4 °C for 30 min and then stained in Permeabilization Wash Buffer (421002; Biolegend, San Diego, CA, USA) with relevant antibodies at 4 °C for 30 min. Foxp3 staining was conducted according to the manufacturer’s instructions for the Mouse Foxp3 Buffer Set obtained from BD Bioscience (San Diego, CA, USA). The following antibodies were used for the studies: APC anti-mouse CD45 (103112), PE anti-mouse F4/80 (123110), PerCP/Cy5.5 anti-mouse F4/80 (123128), FITC anti-mouse CD11c (117306), APC anti-mouse CD206 (141708), APC anti-mouse/human CD45R/B220 (103211), PerCP/Cy5.5 anti-mouse Ly-6G/Ly-6C (108427), FITC anti-mouse CD4 (100406), PerCP anti-mouse CD8a (100732), AlexaFluor 647 anti-mouse/rat/human Foxp3 (320014), PE anti-mouse/human CD44 (103008), and APC anti-mouse CD62L (104412) from Biolegend (San Diego, CA, USA), and PE-Cy7 anti-mouse CD11b (552850) from BD Bioscience (San Diego, CA, USA). Flow cytometry data were acquired on MACSQuant^TM (Miltenyi Biotec, Auburn, CA, USA) and analyzed by FlowJo software (v10.5.3).