Mouse anti human cd81 antibody

The total protein was extracted from cells and exosomes using modified RIPA buffer (Thermo Fisher Scientific) and cell debris was removed by centrifugation. Equal amounts of protein (15–20 μg) were then separated on polyacrylamide gels, transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA) and blotted using mouse anti-human CD81 Antibody (BD Biosciences; Cat: 555676). Membranes were developed using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific).

CD33⁺ myeloid cells were isolated and cultured with CFSE-labeled HCV-Exo for 1 h, followed by examination of fluorescence signal with confocal laser-scanning inverted microscope (Leica Confocal Model TCS sp8, Germany). To observe the structure of exosomes, the purified exosomes (P5 preparation) were seeded on carbon-coated Formvar nickel grids (EM Sciences, Hatfield, PA) and left to adsorb onto grids at 4 °C overnight, fixed with EM-grade 2% paraformaldehyde (EM Sciences, Hatfield, PA) and 0.1% glutaraldehyde (EM Sciences, Hatfield, PA) at 4 °C for 1 h, blocked with 2% cold fish gelatin (Sigma, St. Louis, MO)/0.1% bovine serum albumin/phosphate-buffered saline (PBS) for 30 min at room temperature and incubated with 50 µg/ml mouse anti-human CD81 antibody (BD Pharmingen, San Jose, CA) for 2 h at room temperature. After washing with PBS, the exosomes were incubated with 1:40 diluted 12 nm Colloidal Gold-AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature. After washing with PBS, the grids were stained with 1% aqueous uranyl acetate before observation with a Zeiss Auriga 40 operating in STEM mode at 30 kV electron microscope.