Fresh PBMCs were stained with fluorochrome-labeled anti-human CD11c, CD86, HLA-DR, OX40L, 4-1BBL (BD), Lin (Biolegend), CD40, CD45, CD80, CD1c/BDCA1, TLR4 (Beckman), 4-1BBL (Clinisciences), ICOS-L/CD275, TLR9 (eBiosciences), BDCA2, BDCA3 (Miltenyi), and GITRL (R&D systems) antibodies. TLR3 (Abcam), TLR8 (Novus) and TLR9 (eBiosciences) staining were performed using anti-human TLR after surface molecules staining and cell permeabilization. Fresh liver cell suspension (LMNCs) were stained with anti-human CD11c, CD86, HLA-DR (BD), Lin (Biolegend); CD40, CD45, CD80, CD1c/BDCA1 (Beckman), BDCA2 and BDCA3 (Miltenyi) antibodies. Stained cells were then analyzed using LSRII Flow Cytometer and FACSDiva software (BD). Isotype controls were used to discriminate positive cells from nonspecific background staining and dead cells were excluded with Live and Dead cell stain (ThermoFisher). Mean fluorescence intensity (MFI) was analyzed and shown only when the mean percentage of total positive cells was ≥30%. To ensure quality control during the study, we performed a systematic standardization of the fluorescence intensities using cytometer setup and tracking beads (BD).
Ouaguia L., Leroy V., Dufeu-Duchesne T., Durantel D., Decaens T., Hubert M., Valladeau-Guilemond J., Bendriss-Vermare N., Chaperot L, & Aspord C. (2019). Circulating and Hepatic BDCA1+, BDCA2+, and BDCA3+ Dendritic Cells Are Differentially Subverted in Patients With Chronic HBV Infection. Frontiers in Immunology, 10, 112.
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