Bdca4 apc

Blister fluids derived from suction blisters of forearms treated topically with Neosporin cream or control cream were centrifuged for 5 min at 1,500 rpm. The supernatants were harvested for IL-26 measurement by ELISA (Cusabio Biotech). The cellular fraction was analyzed by flow cytometry (BD FACSCalibur) using the following antibodies: CD45-PerCP-Cy5.5 (BD), CD11b-APC or -FITC (eBiosciences), CD15-FITC (BD), Siglec8-PE (Biolegend), CD14-PE (BD), CD3-PE (BD), CD123-PE (BD), and BDCA4-APC (Miltenyi). Neutrophils were identified as CD45⁺CD11b⁺Siglec8⁻CD15⁺, eosinophils as CD45⁺CD11b⁺Siglec8⁺CD15⁺, monocytes as CD45⁺CD11b⁺CD14⁺CD15⁻, plasmacytoid dendritic cells (pDC) as CD45⁺CD11b⁻CD123⁺BDCA4⁺, and T cells as CD45⁺CD3⁺CD11b⁻.

Ex vivo PBMCs were immediately thawed, washed in cold wash buffer (phosphate-buffered saline, 2% fetal bovine serum, and 1 mM ethylenediamine tetraacetic acid). Cells were blocked with wash buffer containing 5% of heat-inactivated human plasma, followed by staining with CD16-FITC (SouthernBiotech), TRAIL-PE or isotype-matched antibody (BD Biosciences), CD3− Pacific Blue (BioLegend), CD107a-eFluor660 or isotype-matched antibody, and CD56-PE.Cy5.5 (eBioscience), and fixed with 2%paraformaldehyde prior to analysis. Cell cultures were stained with the same antibodies as patients' sample but CD56-PerCP.Cy5.5 (BioLegend) was used instead. For TLR3 detection, cells were further permeabilized with BD®Perm reagent and stained with TLR3-PE (BD Pharmingen). PDCs were assessed with the following antibody cocktail: CD4-PE (SouthernBiotech), CD11c-PE.Cy7 (BD Pharmingen), and BDCA4-APC (Miltenyi). About 1 × 10⁵ events in the lymphocyte gate were acquired when analyzing patients' samples in FACS Aria IIu. For in vitro samples, 5 × 10⁵ events were acquired in the same gate using BD Accuri C6 or FACS AriaIIu. All samples were analyzed in up to 18 hours after fixation. IL12 (R&D Systems) and IFNα (PBL Interferon Source) were detected in cell culture supernatant or plasma samples by ELISA.