Live dead fixable blue dead cells stain kit

rAAV8-HBV1.3-transduced HBV-carrier mice were characterized by evaluating several aspects of T cell function as demonstrated in Supplementary Fig. 4. For the evaluation of cytokine-producing ability of T cells, 2 million hepatic MNCs were seeded per well into 96-well plates and incubated with 30 ng/mL PMA and 1 μg/mL ionomycin (both ordered from Beyotime) for 4 h in the presence of 5 μg/mL brefeldin A (Biolegend). Cytokine production of T cells was evaluated by surface and intracellular staining using Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manuals. For the analysis of PD-1, LAG-3, and TIM-3 expression on T cells, hepatic and splenic MNCs were used and first incubated with Live/Dead Fixable Blue Dead Cells Stain Kit (Thermofisher) for 5 min. After washing, cells were incubated with antibody cocktails for 20 min at 4 °C in dark. Flow cytometry analysis was carried out on BD FACSymphony A3 (BD Biosciences). Data was analyzed using FlowJo V10.8 software (Tree Star). Fluorochrome-conjugated antibodies used in this study are listed in Supplementary Table 2.

Frequencies of HBsAg-specific MBCs were assessed by flow cytometry. For the preparation of HBsAg probes, HBsAg was first biotinylated using a Biotin Quick Labeling Kit (Friendbio Science) according to the manuals. Biotinylated HBsAg was next conjugated with Brilliant Violet 421-streptavidin (Biolegend) at a molar ratio of 4:1. Splenic MNCs were incubated with HBsAg probes for 20 min, and then stained with Live/Dead Fixable Blue Dead Cells Stain Kit (Thermofisher) for 5 min. After washing, cells were incubated with antibody cocktails for 20 minutes at 4 °C in dark. Flow cytometry analysis was carried out on BD FACSymphony A3 (BD Biosciences). Data was analyzed using FlowJo V10.8 software (Tree Star). Fluorochrome-conjugated antibodies used in this study are listed in Supplementary Table 2.