Phenotypic Characterization of Activated pDCs

Referencing a previous in vitro study [10 (link)], PBMCs were stained with a fluorescent dye and fluorescent conjugated antibodies: BD Horizon Fixable Viability Stain 780 (FVS780) viability dye, PE-Cy7 Mouse Anti-Human CD123 (BD, 560826), BB515 Mouse Anti-Human Neuropilin-1 (CD304) (BD, 566036), and APC Mouse Anti-Human CD86 (BD, 555660) following the manufacturer’s instructions, and then fixed with 4% paraformaldehyde. PBMCs were cultured at 1 × 10⁶ cells/mL in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% human AB serum (Sigma-Aldrich), 2 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Thermo Fisher Scientific), and stimulated in the presence of 10 µg/mL TLR-7/8 agonist R848 (InvivoGen, San Diego, CA, USA) for 4 h. Afterward, the cells were harvested and stained with FVS780, PE-Cy7 Mouse Anti-Human CD123, BB515 Mouse Anti-Human Neuropilin-1 (CD304), and PE Mouse Anti-Human HLA-DR (BD, 556644) as described above. Data were collected using CytoFLEX (Beckman Coulter, Brea, CA, USA) and analyzed using the FlowJo version 10 software (BD). Isotype controls (BD) were used to check the background caused by nonspecific antibody binding. pDCs were defined as CD123+CD304+ subsets, and CD86 and HLA-DR were used as activation markers for pDCs.