Flowcytometry analysis
Recombinant HBsAg (rHBsAg, serotype adr) was purchased from Bioforce (China). Purified rHBsAg and hen egg lysozyme (HEL) were conjugated with AlexaFlour 647 (Thermo Fisher Scientific) and iFlour488 (Abcam, China) to generate HBsAg-AF647 and HEL-iFlour488, respectively. HEL-iFlour488 was used for monitoring nonspecific binding. Murine peripheral blood cells were co-stained with Fixable LIVE/DEAD Dye (Life Technologies, Thermo Fisher Scientific), anti-mouse B220 (clone RA3-6B2, eBioscience) Super Bright 436, HBsAg-AF647 and HEL-iFlour488 in FcR-blocking reagent (Biolegend) at 4°C for 30 minutes. All data were acquired with Attune™ NxT Flow Cytometer (Invitrogen), and then analyzed with FlowJo (TreeStar, Ashland, OR, USA). At least 5 × 105 events were acquired per run.