Fitc labeled anti cd80

After 48 h of stimulation, DCs were harvested with 0.4% trisodium citrate (Merck Millipore, Amsterdam, Netherlands) in PBS supplemented with 4 mg/ml human albumin (Albuman, Sanquin Reagents, Amsterdam, Netherlands). DCs were stained 30 min at room temperature with LIVE/DEAD Fixable near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) and subsequently with either PE-labeled anti-HLA-DR (Clone G46-6) or with FITC-labeled anti-CD80 (Clone L307.4), PE-labeled anti-CD40 (Clone 5C3), APC-labeled anti-CD83 (Clone HB15e) and BV421-labeled anti-CD86 (Clone 2331) (all from BD Biosciences) in the presence of 3 mg/ml human γ-globulin (Nanogam, Sanquin, Amsterdam, Netherlands). Samples were measured with BD FACSCanto II and analyzed using Flowjo VX (Beckton, Dickinson & Company, Ashland, OR, United States). Gating strategy involved selection of DCs on FSC/SSC, single cells and viable cells.

FITC-labeled anti-CD80, PE-labeled anti-CD86, PE-Cy5-labeled anti-CD83, and APC-conjugated anti-CD209 and anti-CD95 antibodies were obtained from BD Pharmingen (San Diego, CA, USA). Dead cells were stained using the Live/Death detection kit with a near-infrared dye (Invitrogen, Carlsbad, CA, USA). The samples were analyzed using CyAn ADP Analyser (Beckman Coulter, Brea, CA, USA), and the data were analyzed using FlowJo version 9.2 (Tree Star Inc., Ashland, OR, USA).