Pe conjugated anti icosl

Briefly, 1×10⁶ splenocytes were incubated in 100 μl of PBS. The cells were then stained with a mixture of PE- and FITC-conjugated mAbs for 30 min, washed twice, then fixed with 4% paraformaldehyde in PBS. The analysis was performed on a FACS Calibur Flow Cytometer (BD Biosciences). All procedures were performed on ice until the time of analysis. The mAbs for flow cytometry were purchased from eBioscience (San Diego, USA) and included FITC-conjugated anti-CD4, FITC-conjugated anti-CD19, PE-conjugated anti-ICOS, PE-conjugated anti-ICOSL, PE-conjugated anti-RORγtand PE-conjugated anti-IL-17R.

Lymphomononuclear cells from the livers, spleens and peripheral blood were incubated with fluorochrome-labeled antibodies at 5×10⁵ / tube for 30 min at 4°C, and characterized using a FACS Canto II cytometer (BD Biosciences). For the intracellular cytokine staining, Caltag^TM, Fix&Perm^® reagents (Invitrogen, Carlsbad, CA, USA) were used following the manufacturer's instructions. The following antibodies were used: fluorescein isothiacyanate (FITC)-conjugated anti-CD4, FITC-conjugated anti-CXCR5, FITC-conjugated anti-CD19, Phycoerythrin (PE)-conjugated anti-PD-1, PE-conjugated anti-ICOS, PE-conjugated anti-PDL-1, PE-conjugated anti-ICOSL, PE-conjugated anti-IL-21R, allophycocyanin (APC)-conjugated anti-CD4 (eBioscience, USA), PE-conjugated anti-IL-21 (BD Biosciences, USA) and FITC-, APC- or PE-conjugated isotype antibodies (eBioscience, USA).
Lymphomononuclear cells from the livers and spleens were incubated with HBc-derived peptides HBc1-20 (1 mg / ml)at room temperature for 10 min. HBc-derived peptides HBc1-20 was purchased from Sangon Biotech (Sangon, Shanghai, China). The cells were then washed twice with PBS containing 1% BSA (BD Biosciences), and incubated with anti-CD4, anti-CXCR5 for 30 min at 4°C, and characterized using a FACS Canto II cytometer.