Pe anti mouse cd206 antibody

To measure the percentage of M2-like TAMs, excised tumours from various tumour models were prepared for single-cell suspension after in vivo fluorescence imaging by the IVIS Spectrum imaging system. The samples were stained with PerCP/Cy5.5 anti-mouse CD45 antibody (103132, Biolegend, clone number: 30-F11, Dilution 1:100), FITC anti-mouse/human CD11b antibody (101205, Biolegend, clone number: M1/70, Dilution 1:200), PE/Cy7 anti-mouse F4/80 antibody (123114, Biolegend, clone number: BM8, Dilution 1:100), and PE anti-mouse CD206 antibody (141706, Biolegend, clone number: C068C2, Dilution 1:40) according to the manufacturer’s instructions. The proportions of CD11b⁺F4/80⁺CD206⁺ macrophages were obtained by flow cytometry, and the correlation analysis between M2-like macrophage content and the ratiometric signal was measured by GraphPad Prism 8 software.

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).

CII-3 was produced by Kunming SINOWAY Natural Pharmaceuticals Co., Ltd., Kunming, China, provided by Professor Liu Guang-ming of Dali University.
RAW 264.7 cells, DMEM, and RPMI 1640 were procured from Procell Life Science & Technology Co., Ltd. Wuhan, China. In addition, the Ana-1 cell line was obtained from Professor Yang Guo-ping of Dali University. The following reagents were utilized in the study: lipopolysaccharide (LPS) (Solarbio Science and Technology Co., Ltd., Beijing, China); Interferon-γ (IF-γ) (Peprotech Inc., East Windsor, NJ, USA); Interleukin-4 (IL-4) (Peprotech Inc., NJ, USA); MTT (Solarbio Science and Technology Co., Ltd.); RNeasy Isolation Reagent method, HiScript II qRT SuperMIX for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China), ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China); ELISA kits (Shanghai Enzyme Biotechnology Co., Ltd., Shanghai, China); PE anti-mouse CD206 antibody (BioLegend, San Diego, CA, USA) used for flow cytometry detection; CD206 antibody (Affinity Biosciences, Cincinnati, OH, USA) used for immunofluorescence; FITC-conjugated goat Anti-Rabbit IgG (H+L) (Affinity Biosciences, Cincinnati, OH, USA); DAPI (Solarbio Science and Technology Co., Ltd.); as well as PCR primers (Bioengineering (Shanghai) Co., Ltd., Shanghai, China)