Cd80 pe cy5

For analysis of the phenotype of BDCA-1⁺/BDCA-3⁺ myDC, cells were stained with CD11c-Alexa Fluor 700 (BD Biosciences, clone B-ly6), CD1c-Brilliant Violet 510 (BD Biosciences, clone F10/21A3), CD141-PE/Cy7 (Invitrogen, clone JAA17), CD274-PE-CF594 (BD Biosciences clone MIH1), CD86-Brilliant Violet 421 (BD Biosciences, clone 2331 (FUN-1)), CD83-PE (BD Biosciences, clone HB15e), CD40-APC (BD Biosciences, clone 5C3), CD80-PE/Cy5 (BD Biosciences, clone L307.4), HLA-ABC-FITC (BD Biosciences, clone G46-2.6), Zombie Yellow (Biolegend) for 20 minutes at 4°C. After washing, cells were resuspended in PBS/0.5%BSA and acquired on a BD LSR Fortessa instrument. Data analysis was performed using FlowJo software. The gating strategy was as follows: cells were first gated on FSC/SSC characteristics, followed by gating on single cells. Next, dead cells were excluded and subsequently we gated on the CD11c⁺ population. On this gate, CD1c⁺ CD141^- cells were identified as BDCA-1⁺ myDC and CD1c^- CD141⁺ cells as BDCA-3⁺ myDC. Subsequently, we evaluated for each myDC subtype the expression of HLA-ABC, CD83, CD274/PD-L1, CD80, CD40 and CD86.

For the differentiation and polarization of isolated human monocytes into M1 and M2 macrophages, cells were cultured for 7 days at 37 °C in a 5% CO₂ atmosphere in complete medium composed of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution in the presence or absence of different cytokines. Monocytes were seeded in 96- or 6-well culture plates at 2 × 10⁵ cells/well or 1 × 10⁶ cells/well, respectively, and the following cytokines were added for 7 days: 50 ng/mL GM-CSF (for M1 polarization) or 50 ng/mL M-CSF (for M2 polarization). At day 6, 100 ng/mL LPS was added to M1 polarized macrophages and 20 ng/mL IL-4 was added to M2 polarized macrophages. Seven days later, cells were detached from plates and stained with a cocktail of conjugated antibodies as follows: 10 µL CD14-FITC, 5 µL CD16-APC-H7, 5 µL CD80-PE-Cy5, 5 µL CD163-APC, and 5 µL CD200R-PE from BD Biosciences. Data acquisition was performed on 10,000 cells with constant PMT values on an FACS Canto II cytometer (BD Biosciences). Data analysis was carried out using Diva software (BD FACSDiva v9.0software, BD).