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4 protocols using anti mouse cd16 cd32 antibody clone 93

1

Quantifying Fc Receptor Expression on Macrophages

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RAW264.7 cells were co-incubated with IgG-opsonized sheep erythrocytes. Following blocking with FcR antibody (anti-mouse CD16/CD32 antibody, clone 93; Biolegend, Dedham, MA, USA), CR Mac-1, CR4 (CD11c/CD18) and FcR FcγI on RAW264.7 cells were probed using phycoerythrin (PE) labeled anti-mouse CD11b antibody (clone M1/17), anti-mouse CD11c antibody (clone N418) and anti-mouse CD64 antibody (clone X54-5/7.1), respectively (all from Biolegend). FcR FcγII/III was detected with PE-labeled anti-mouse CD16/32 antibody (clone 93). Then cells were subjected to flow cytometry analysis. Sheep erythrocytes were gated out by forward scatter and negative CD11b expression.
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2

Cytokine and Surface Marker Analysis of Murine Bone Marrow-Derived Dendritic Cells

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Murine BMDCs were seeded at a density of 1 × 105 per well in a 96-well flat-bottomed culture plate. Cells were stimulated with 0.1 μg/mL ODN1826, 1 μg/mL CpG K3, 1 or 10 μg/mL CpG D35, 1 μg/mL ODN1585, 1 μg/mL poly(I:C), or 10 μg/mL c-di-GMP with or without AS1411 (1 or 5 μM), CRO (1 or 5 μM), MS-3 (50 μg/mL), isotype control antibody (50 μg/mL), or 0.4 μg/mL H-151 for 24 h at 37 °C. Supernatants were subjected to ELISA to measure IL-6 (BioLegend), IL-12 p40 (BioLegend), IL-12 p70 (BioLegend), and IFN-α (InvivoGen) secretion in accordance with the manufacturer’s instructions. To check the surface levels of CD86 on DCs, we incubated the cells with anti-mouse CD16/CD32 antibody (clone: 93, catalog numbers: 101302, dilution 1/200; BioLegend), APC-Cy7-labelled anti-CD11c antibody (clone: N418, catalog numbers: 117324, dilution 1/200; BioLegend), APC-labelled anti-PDCA-1 antibody (clone: 927, catalog numbers: 127016, dilution 1/200; BioLegend), and PE-labelled anti-CD86 antibody (clone: GL-1, catalog numbers: 105008, dilution 1/200; BioLegend). DCs were separated into the following subsets: CD11c+ PDCA-1- cDCs and CD11c+ PDCA-1+ pDCs. The cells were then analyzed using flow cytometry.
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3

Murine Brain Tumor Immune Cell Isolation

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Single cells from the murine brain earing GL261 tumors were isolated using a mouse tumor dissociation kit (Miltenyi Biotec, Germany) and a gentleMACS Octo dissociator with heaters (Miltenyi Biotec). Myelin was depleted using 25% BSA density gradient centrifugation for 20 min at 2,600 rpm and subsequently removing the myelin rim on top of the BSA gradient. CD45+ cells were enriched using mouse CD45 MicroBeads (Miltenyi Biotec) following the manufacturer’s instructions. Unspecific Fc receptor binding in all single-cell suspensions was then blocked by using anti-mouse CD16/CD32 antibody (clone 93, BioLegend, San Diego, CA, USA), and were then stained using fluorochrome-conjugated antibodies (Table S2). All antibodies were diluted 1:100 from stock concentration in Brilliant Violet stain buffer (BD Biosciences). CountBright absolute counting beads (Thermo Fisher Scientific) were added to calculate absolute numbers of immune infiltrates, and samples were acquired in an LSRFortessa (BD Biosciences). Data were analyzed using FlowJo version 10.7 (FlowJo) or SPICE.37 (link)
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4

Multiparameter Flow Cytometric Analysis

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Cells were stained using a live-dead dye (Supplementary Tables 4 and 5) following the instructions of the manufacturer. Unspecific Fc receptor binding in all single-cell suspensions was blocked by using anti-mouse CD16/CD32 antibody (clone 93, Biolegend, San Diego, CA, USA). Cells were stained for the markers of interest using fluorochrome-conjugated antibodies (Supplementary Tables 4 and 5). All antibodies were diluted from stock concentration according to the ratios reported in Supplementary Tables 4 and 5. For staining of FoxP3, the FOXP3 Fix/Perm Buffer Set (BioLegend) was used following the instructions of the manufacturer. For intracellular cytokine staining, the eBioscience™ Invitrogen™ Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA) was used following the instructions of the manufacturer. Samples were run on FACSCanto II, LSR Fortessa (BD BioSciences, San Jose, CA, USA) (data was collected using BD FACSDiva 8.0.2) or CytoFLEX LX (Beckman Coulter, Brea, CA, USA) (data was collected using CytExpert 2.4); alternatively, the cells were sorted directly into RLT lysis buffer (Qiagen) using FACS AriaIII (BD BioSciences). Data were analyzed using FlowJo version 10.5.3 (FlowJo LLC, Ashland, OR, USA) or Cytosplore version 2.2.156 (link),57 (link). Gating strategies used in this paper can be found in Supplementary Figs. 12 and  13.
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