Phenotyping and ZIKV Infection of pDCs

The phenotype of pDCs was assessed with the following primary mAbs (BD Horizon): CCR7-FITC (clone 3D12), CD83-PE-Cy7 (clone HB15e), CD86-PE-Cy5 (clone 233), HLA-DR-APC-H7 (clone L243), and CD123-PerCP-Vio 700 (clone AC145). CD303-PE (clone REA693) was purchase from MACS. Cells were fixed with 4% PFA for 20 min, stained for 30 min at 4°C and washed before being subjected to FACS analysis. ZIKV infectivity was assessed with the mouse anti-pan flavivirus envelope protein mAb 4G2 (RD Biotech). A ZIKV-specific polyclonal antibody targeting the envelope protein and the non-structural protein 1 (20 (link)) or the mouse anti-dsRNA IgG2a mAb J2 (Scicons) as indicated. Cells were fixed with 4% PFA for 20 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 in PBS for 4 min at RT. Fixed cells were stained overnight at 4°C using 4G2 or J2 (1:500) in PBS-BSA. Then, cells were stained 20 min at RT with secondary antibody donkey anti-mouse Alexa Fluor 488 IgG (1:1000, Invitrogen) or donkey anti-mouse Cy3 (1:500, Jackson immunoResearch) in PBS-BSA, and washed before being subjected to FACS analysis. At least, 5,000 events were acquired using Cyan cytometer (Beckman Coulter). Stained cells were analyzed using FlowJo software (Tree Star, Inc., Ashaland, OR). pDCs survival was assessed using the 7-AAD assay as previously described (21 (link)).