Anti cd11c alexafluor700

Characterization of immune cell subsets in the liver was performed essentially as described previously^{21 (link)}. The individual samples were analyzed with a LSRII/Fortessa flow cytometer (BD Biosciences) and the FlowJo software Vx (Treestar). All indicated antibodies and reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using cytometer setup and tracking beads (BD). Single cell suspensions were created using the Miltenyi Liver Dissociation Kit (No. 130-105-807) and the GentleMACS isolator (Miltenyi) using standard protocols. The following antibodies were used: anti-CD3-PE-CF594, anti-CD4-V500, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326 (EPCAM)-BV711, anti-Ly6C-PerCP-Cy5.5 (all from BD), anti-CD8-eFluor650, anti-CD11b-eFluor605NC (eBioscience), anti-CD45-VioBlue, anti-CD49b-PE, anti-MHC-II-APC (Miltenyi), anti-F4/80-PE-Cy7, anti-Ly6G-APC-Cy7 (Biolegend). A gating strategy is provided in the supplementary material and methods (Fig. S1).

Upon treatment, BDCA-1⁺ myDCs were seeded in a 96-well round bottom plate at a density of 150,000–200,000 cells per well. BDCA-1⁺ myDCs were treated with either active T-VEC (MOI 10), heat-inactivated T-VEC (MOI 10; heat-inactivation: 15 min 65 °C, 1 min 100 °C) or conditioned medium, i.e., SN of dying 624-mel and 938-mel 24 h and 48 h after treatment with T-VEC (MOI 1). Untreated BDCA-1⁺ myDCs served as a negative control. As a positive control, BDCA-1⁺ myDCs were treated with a mix containing ssRNA fragments derived from HIV-1 long terminal repeat and protamine sulfate (PS/LTR). After overnight incubation, cells were harvested and stained with anti-CD11c-AlexaFluor 700 (BD Biosciences, 561352), anti-CD1c-BV510 (BD Biosciences, 742747), anti-CD80-PerCP-eFluor710 (ThermoFisher Scientific, 46-0809-42), anti-CD86-BV421 (BD Biosciences, 562432), anti-CD40-APC (BioLegend, 334310), anti-CD83-PE (BD Biosciences, 556855), anti-CD274-PE-CF594 (BD Biosciences, 563742) and anti-HLA-ABC-FITC (BD Biosciences, 557348) for 20 min at 4 °C and in the dark. Cells were acquired on the flow cytometer (BD LRS Fortessa) and data were analyzed with with FlowLogic software (Miltenyi Biotec, Version 7.3).

Characterization of immune cell subsets was performed essentially as described previously (20 (link)). Samples were acquired with a LSRII/Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software Vx (Treestar). All antibodies and secondary reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking beads (BD). For characterization of immune cell subsets, the following antibodies were used: anti-CD3-PE-CF594, anti-CD4-BV711, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326-BV711, anti-Ly-6C-PerCP-Cy5.5, anti-NK1.1-BV510 (all from BD Biosciences), anti-CD8-BV650, anti-CD11b-BV605, anti-F4/80-PE-Cy7, anti-GITR-FITC, anti-Ly-6G-APC-Cy7 (from BioLegend), anti-CD31-PE-Cy7, anti-CD117-APC-eFluor780 (from eBioscience), anti-CD45-VioBlue, and anti-HLA-DR-APC (from Miltenyi).