Fixable viability dye efluor 780 fvdye

Monoclonal antibodies (Abs), specific for cluster of differentiation (CD)1a, CD14, CD38, CD86, CD83, HLA-DR, CD40, IgG1, and IgG2a (BD Bioscience, San Diego, CA, USA), were directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). To exclude dead cells from the analysis, Fixable Viability Dye eFluor®780 (FvDye) (eBioscience, San Diego, CA, USA) was used. For immunoblotting analysis, rabbit anti-STING (Cell Signaling, Danvers, MA, USA # 2775), anti-IRF3 (Santa Cruz, Santa Cruz, TX, USA # sc-9082), anti-IRF7 (Santa Cruz, # sc-9083), anti-STAT1 (BD Bioscience, # 610186), anti-phospho STAT1 (Cell Signaling Technology, Leiden, The Netherlands, # 7649), anti-STAT2 (BD Transduction Laboratories, # 610188), anti-phospho STAT2 (R&D Systems, Minneapolis, MN, USA, MAB2890), mouse anti-actin (Sigma-Aldrich, St. Louis, MO, USA #A0483), and horseradish peroxidase-conjugated secondary antibody anti mouse (Santa Cruz, # sc-2005) and anti rabbit (Santa Cruz, # sc-2004) were used. For phagocytosis and phagosomal acidification experiments, cytochalasin D 5 μM (Sigma-Aldrich, # C8723) and chloroquine 2 μM (Sigma-Aldrich, # C6628) were used.

Monoclonal Ab specific for CD14, CD19, CD38, CD27, PD-L1, CD80, CD86, IL-T7, HLA-DR, CD123 as well as IgG1 or IgG2a isotype controls were purchased from BD Biosciences (San Diego, CA, USA), BDCA2 from Biolegend (Fell, DE), BDCA4 from Miltenyi Biotech and IgM by Jackson Laboratories (Bar Harbour, Maine, USA). To establish viability of cells and to exclude dead cells from flow cytometry analysis Fixable Viability Dye eFluor780 (FvDye) (eBioscience, San Diego, CA, USA) was used as previously described [68 (link)]. Briefly, cells (10⁵ for PBMC and 5x10⁴ for isolated pDC) were incubated with monoclonal Abs at 4°C for 30 min and then fixed with 2% paraformaldehyde before analysis on a Gallios cytometer (Beckman Coulter). Data were analyzed by Kaluza software (Beckman Coulter). The expression of cell surface molecules was evaluated using the median fluorescence intensity (MFI) after subtraction of the values of the isotype Ab controls. Only cells present in the viable cell gate were considered for the analysis.