Fix perm reagents

Stimulated whole blood was stained for flow cytometric study to identify cytokine-producing cells. After incubation, 100 µL blood was stained with PerCP-Cy5.5–conjugated anti-human CD4 monoclonal antibodies (clone: OKT4, Cat.: 85-45-0048-42) at room temperature for 20 min. The sample was then treated with an equal volume of Fix-Perm reagent A for 15 min, and then washed with pre-cooled washing buffer. Subsequently, Fix-Perm reagent B was added for further permeabilization for intracellular staining and erythrocyte lysis. The sample was incubated for 20 min together with PE-conjugated anti–IFN-γ monoclonal antibodies (clone: 4S.B3, Cat.: 85-11-7319-82), FITC-conjugated anti–IL-17A monoclonal antibodies (clone: eBio64D, Cat.: 85-11-7179-42), and eFluro680-conjugated anti–IL-22 monoclonal antibodies (clone: 22URTI, Cat.: 85-50-7229-42). Isotope controls were used for each staining procedure as negative controls and for fluorescence compensation. All antibodies were from eBioscience, San Diego, CA, USA. Fix-Perm reagents were from Invitrogen (Carlsbad, CA, USA). All samples were washed and collected using BD Accuri C6 Flow Cytometer. Data were analyzed with FlowJo 7.6.

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.

Cells were washed twice with flow cytometry buffer (PBS, 0.5% FBS) and then incubated for 45 min at 10 °C before being labeled with antibodies in flow cytometry buffer. After 3 washes, cells were resuspended in FACS buffer and analyzed by flow cytometry on a Beckton Dickinson (BD) FACSCanto™. Data were analyzed with FlowJo software (Treestar Inc., CA).
For NFAT translocation analysis, T cell nuclei were purified with the Nuclei isolation Kit (NUC201 Sigma-Aldrich), fixed, and permeabilized with the fix/Perm reagents (from 00-5523-00 eBioscience) and then stained with AF488-conjugated anti-NFAT mAb. After 3 washes, nuclei were resuspended in FACS buffer and analyzed by flow cytometry on a BD FACSCanto™. Data were analyzed with FlowJo software (Treestar Inc., CA).