Sa pe cy5

From a leukapheresis preparation from healthy humans with the HLA-A2 haplotype (Biological Specialty Corporation, 213-15-04), human PBMCs were obtained by Ficoll-Hypaque (Sigma-Aldrich, GE17-1440-02) density gradient centrifugation.²⁴ The PBMCs isolated from uninfected human donors or naïve sheep were cultured in 100 mm culture dishes (BD Biosciences, 353003) for 1 h at 37°C in AIM V media (Thermo Fisher Scientific, 12055–091) with 2.5% autologous heat inactivated plasma. Following culture, the non-adherent cells were removed and adherent cells harvested and seeded into 96 well v-bottom plates (Corning, 3894) at 2 × 10⁵ cells/well. All remaining steps were performed at 4°C. Cells were incubated for 1 h with 1, 5, 20, 50 µg/mL C-HBV diluted with PBSB (Dulbecco’s phosphate buffered saline, Thermo Fisher Scientific, 14190–250 containing 0.1% (w/v) bovine serum albumin (BSA)). Protein binding was detected by incubating cells with rat anti-mouse IgG₁-biotin (BD Biosciences, 553441) in PBSB for 20 min, followed by streptavidin phycoerythrin cyanine-5 (SA-PE-Cy5;BD Biosciences, 554062) for 20 min. Cells were re-suspended in PBSB containing 2% paraformaldehyde (PF) and surface binding of C-HBV assessed by flow cytometry.