Apc anti mouse f4 80

To analyze M1-like macrophages, BMDMs were stained with PerCP/Cyanine5.5 anti-mouse/human CD11b (101228; BioLegend), APC anti-mouse F4/80 (123116; BioLegend), and Alexa Fluor 488® anti-mouse CD86 (105017; BioLegend). For intracellular iNOS staining, BMDMs after Fc-blocking were stained with PerCP/Cyanine5.5 anti-mouse/human CD11b (101228; BioLegend) and APC anti-mouse F4/80 (123116; BioLegend) at 4°C for 30 min, fixed and permeabilized with BD Cytofix/Cytoperm Kit (554714; BD Biosciences) at 4°C for 20 min. After washing with Perm/Wash buffer (BD Biosciences), the cells were stained with Alexa Fluor 488 anti-iNOS (53-5920-80; Invitrogen) at 4°C for 30 min. The stained cells were resuspended in 2% paraformaldehyde and analyzed using the NovoCyte 2060 R (ACEA Biosciences, USA), using FlowJo software ver 10.7.2 (BD Biosciences)

We used mouse J774 cells, non-obese diabetic, severe combined immunodeficient, common gamma chain knockout mouse (NOD-SCID-Gamma, or NSG) macrophages, and human macrophages for our experiments. J774 macrophages were activated 24 hours before phagocytosis assays using 100 ng/ml recombinant mIFNγ (eBioscience). Cancer cells were either GFP+ or labelled with carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and were incubated with 10 μg/ml of CD47 blocking reagents, isotype controls, or tumour-targeting antibodies for 30 minutes. Macrophages were then co-cultured in non-adherent plates with cancer cells at a 1:1 (J774) or 1:2 (NSG, human macrophages) ratio. Phagocytosis was analysed by flow cytometry after 2 hours. Mouse macrophages were identified with PE/Cy7- or allophycocyanin (APC)-anti-mouse F4/80 (Biolegend), and human macrophages were identified with APC-anti-human CD206 (Biolegend). Phagocytosis was quantified as the percentage of F4/80+ or CD206+ cells that engulfed CFSE+/GFP+ tumour cells per total F4/80+ or CD206+ population. For soluble factor assays, Raji cells and J774 macrophages were suspended in supernatant harvested from cultured M14 melanoma cells titrated with new IMDM medium for the duration of the assay.

RAW264.7 cells were incubated in RPMI 1640 medium with 10 μg/ml SCAT-EXOs, 10 μg/ml PVAT-EXOs or equal amounts of PBS for 24 h, and then 50 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich) was added for another 24 h. After being washed twice with cold PBS, the cells were harvested and incubated for 30 min at 4°C in the dark with antibodies including APC-anti-mouse F4/80 (Biolegend, CA, USA), PE-anti-mouse CD80 (Biolegend) and FITC-anti-mouse CD206 (Biolegend). The cells were analysed by flow cytometry (BD Pharmingen, NJ, USA) and FlowJo 7.5 (FlowJo, OR, USA).