Anti bdca1 pe

Blood DCs from healthy donors were separated by double centrifugation procedures with Ficoll-Hypaque and Percoll 51% (GE Healthcare). After washes, blood DCs and previously differentiated Mo-DCs were incubated with LPS (100 ng/mL); poly-mixin B (10 mg/mL); LPS þ polymixin B (100 ng/mL þ 10 mg/mL); gDE7 þ polymixin B (3 mg/mL þ 10 mg/mL); gD þ polymixin B (2.25 mg/mL þ 10 mg/mL); or E7 þ polymixin B (0.75 mg/mL þ 10 mg/mL). The cells were incubated with the Golgi Plug (10 mg/ mL; BD Biosciences) reagent for 6 hours and stained with anti-HLA-DR V500 (cat: 561224), anti-CD11c V450 (cat: 560369), anti-CD14 APC (cat: 555399), anti-BDCA1 PE (Miltenyi Biotec; cat: 130-090-508), and anti-BDCA3 PE-Vio770 (Miltenyi Biotec; cat: 130-100-217) mAbs. Cells were fixed/permeabilized and stained with the anti-TNFa-FITC (cat: 554512) mAb. The percentages of TNFa þ cells over all Mo-DCs, BDCA3 þ , or BDCA1 þ DCs were determined. Unless stated differently, all antibodies were from BD Biosciences. Samples were acquired on a LSR Fortessa flow cytometer (BD Biosciences) and analyzed using the FlowJo software (Tree Star).