Lineage cocktail 1 fitc

Dendritic cells were characterized using the six-color flow cytometry assay as we described previously (17 (link)). Briefly, 100 µl of WB were incubated with the following antibodies: CD45-V500, lineage cocktail1-FITC, HLA-DR-APC-Cy7, CD123-PECy5, all from BD Biosciences and CD11c-PECy7 (Beckman Coulter, Marseille, France). Absolute numbers of DCs were determined using BD Trucount™ Tubes (BD Biosciences). Samples were analyzed using a BD FacsCanto II analyzer with DIVA software (BD Biosciences, Le Pont de Claix, France). The whole tube was acquired to ensure a minimum of 1,000 events in the Lin⁻ HLA-DR⁺ (total DCs) gate for each sample.

Evaluation of MDSC percentage was accomplished with 0.5 × 10⁶ PBMCs stained with anti-CD15 FITC, anti-CD33 PE, anti-HLA-DR PERCP, cocktail of antibodies anti-CD3, -CD56, -CD19 (Lin) APC, anti-CD14 APC-H7, anti-CD11b PE-C7, anti-CD16 Pacific Blue (BD Biosciences). The frequency of hematopoietic progenitors was evaluated on 1 × 10^∧5 viable cells by using Lineage cocktail 1 FITC, anti-CD34 APC, anti-CD45 V500, 7AAD (BD Biosciences), and anti-CD38 PE-Vio770 (Miltenyi Biotec). Intracellular flow cytometry was performed by using anti-CD3, anti-CD8, and anti-IFN-γ (BD Biosciences, USA). Acquisition of 100,000 events was performed in the leukocyte-gated population on FACS CANTO II and analyzed with FACS DIVA software (BD Biosciences).

Human blood DCs were isolated from PBMCs using leukapheresis products. DCs were first enriched using human Pan-DC Pre-Enrichment Kit (StemCell Technologies) and then stained with fluorochrome-conjugated specific antibodies including Lineage cocktail 1-FITC (BD, dilution: 1:10), CD11c-V450 (B-ly6, BD, dilution: 1:50), CD1c/BDCA1-PerCp-Cy5.5 (L161, Biolegend, dilution 1:25), HLA-DR-APC-eFlour780 (LN3, eBioscience, dilution: 1:50), CD303/BDCA-2-PE (AC144, Mitenyi Biotec, dilution: 1:25) and CD141/BDCA3-APC (AD5-14H12, Mitenyi Biotec, dilution: 1:25). After washing with PBS, DCs were sorted as Lineage-HLA-DR+CD11c+ cells differentially expressing CD1c and CD141 using FACSAria II with Diva software (BD).