Cd80 apc h7 l307.4

DC phenotypes were determined by analysis of median fluorescence intensities of surface marker expression measured on a FACS Canto II flow cytometer (BD Biosciences). Cells were harvested and resuspended in FACS buffer (PBS, 1% BSA, 2 mM EDTA) before staining (30 min, 4 °C, dark) using the following antibodies: CD1a-BV421 (HI149), CD40-FITC (SC3) and CD86-PE (IT2.2) (eBioscience), CD80-APC-H7 (L307.4) and PD-L1-PE-Cy7 (MIH1) (BD Biosciences), HLA-DR-APC (LN3, Invitrogen), PD-L2-APC-Vio770 (Miltenyi) as well as LD (eBioscience Fixable Viability Dye eFluor 780 and eFluor 506). Analysis of the median fluorescence intensity of the mentioned markers within the living CD1a⁺ moDC population was performed with FlowJo Software. Cytokine ELISA in supernatants of infected cells was performed according to the manufacturer’s instructions: IL-1β (R&D) and IL-6, IL-8, IL-12, TNF-α (all Peprotech).

First, supernatant was removed and stored for CBA analysis, and then wells were carefully washed with PBS and 10 mM EDTA/PBS was added for 5 min at 37°C. Cells were subsequently gently harvested using a cell lifter. For the FACS staining, macrophages were blocked with the FcR blocking reagent, human (Miltenyi) prior to surface staining for CD80 APC-H7 (L307.4, BD), CD86 FITC (FM95, Miltenyi), HLA-ABC PE (REA230, Miltenyi), HLA-DR APC (AC122, Miltenyi), CD163 PerCPCy5.5 (GHI/61, BD), CD206 APC (19.2, BD), CD209 PerCPCy5.5 (DCN46, BD), and PD-L1 PE-Cy7 (REA1197, Biolegend) for 30 min in the dark and on ice. Directly before measuring the sample on the flow cytometer, 30 ng DAPI (Sigma-Aldrich) was added. To control for background staining, cells stained only with 30 ng DAPI were used. All flow cytometric analyses were performed with BD Canto II and data were analyzed with FlowJo 10.1r5 64-bit software.