Apc conjugated cd19

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by density gradient centrifugation on Ficoll-Paque PLUS (Cytiva). The isolated PBMCs were then analyzed with an 8-color flow cytometer (FACSCanto™II, BD Biosciences, CA, USA) after labeling with the following anti-human fluorochrome-conjugated antibodies: FITC-conjugated CD38 (clone HIT2); APC or FITC-conjugated PD-1 (clone EH12.2H7); FITC-conjugated CD45RA (clone HI100); PE-conjugated CD24 (clone ML5); PE-conjugated ICOS (clone C398.4A); PE-conjugated CCR6 (clone G034E3); PerCP-conjugated CD3 (clone UCHT1); PE/cyanine7-conjugated CD27 (clone O323); PE/cyanine7-conjugated CD4 (clone OKT4); APC-conjugated CD19 (clone HIB19); APC-conjugated CD45RO (clone UCHL1); APC-conjugated CXCR3 (clone G025H7); and Brilliant Violet 421-conjugated CXCR5 (clone J252D4) (all antibodies were from BioLegend, CA, USA). Cell viability was assessed using a live/dead assay (Fixable Viability Dye eFluor 780; eBioscience). All data were collected and analyzed using FlowJo software. The gating strategies of the isotype-matched IgG control for specific cell populations are shown in the supplementary figure (Figure S1).

PBMCs from HCs and patients with AD were resuspended in culture medium (at 2 × 10⁶ cells/mL) in 48-well flat-bottom plates and stimulated for 48 h at 37 C with CpG (TLR9 ligand, 10 μg/mL; In vivogen) and CD40 ligand (CD40L, 1 μg/mL; BioLegend). Phorbol 12-myristate 13-acetate (PMA, 20 ng/mL; Sigma-Aldrich), ionomycin (1 μg/mL, Sigma-Aldrich), and brefeldin A (BFA, 5 mg/mL, BioLegend) were added for the last 5 h. Next, the cells were harvested and washed twice with phosphate buffer solution (PBS). Single-cell suspensions were then stained for 20 min on ice with predetermined optimal concentrations of APC-conjugated CD19 (BioLegend). Stained cells were washed twice with PBS before fixation and permeabilization in Fixation and Permeabilization Buffer (BD Biosciences). Finally, cells were stained for 30 min with PerCP-conjugated IL-10 (clone JES3-9D7, BioLegend).