Anti cd81

Huh-7.5 cells were pre-chilled at 4 °C for 1 h and then treated with HCVcc (MOI = 0.01) in the presence of various concentrations of the test compound at 4 °C for an additional 3 h. Following virus adsorption, the cell monolayers were washed twice with ice-cold PBS before fixation with pre-chilled 4% paraformaldehyde (Sigma). Detection of cell surface-bound virus through ELISA was performed as previously reported27 (link) using the anti-HCV E2 primary antibody (1: 20,000; Austral Biological, San Ramon, CA, USA), followed by a goat anti-mouse IgG conjugated with horseradish peroxidase secondary antibody (1: 36,000; Invitrogen), and developed with a TMB (3,3′,5,5′-tetramethylbenzidine) Two-component Microwell Peroxidase Substrate Kit (KPL; Gaithersburg, MD, USA). Results were measured with an ELx800 Microplate reader (Instrument, Inc.; Winooski, VT, USA) at 450 nm and data were expressed as fold change of A₄₅₀nm readings. Anti-CD81 (10 μg/ml; BD Biosciences, San Diego, CA, USA) treatment and the Huh-7-derived S29 cells, which lack CD8135 (link), were included as positive controls, whereas DMSO (0.5%) treatment served as negative control. A schematic for the procedure is depicted in Fig. 6a.

1 × 10⁶ HepG2 or HEK293T cells were detached from the cell culture plate using Accutase (Stemcell technologies #07920) and washed twice in PBS + 2%BSA. Cells were then incubated in 100 µl of PBS + 2%BSA + Anti-CD81 (BD Biosciences #555675, clone JS-81, 1/200 dilution) for 30 min at 4 °C. Cells were then washed three times in PBS + 2% BSA and incubated in 100 µl of PBS + 2 %BSA + anti-mouse FITC (Biolegend # 406001, 1/2000 dilution) for 30 min at 4 °C in the dark. Cells were then washed three times in PBS + 2%BSA and fixed with 4% of paraformaldehyde (PFA) for 15 min and washed in PBS + 2%BSA before flow cytometry analysis on a BD FACSCanto II.

The StemPro Differentiation Kit (Life Technologies) was used according to the manufacturer’s recommendations to determine the capacity of hMSCs to undergo adipogenic or chondrogenic differentiation. Characterization of hMSCs and hMSC-microvesicles was performed by fluorescence-activated cell sorting (FACS) utilizing a FACSCalibur™ sorter (BD Biosciences) and analyzed by CellQuest software (BD Biosciences). FITC-conjugated antibodies (anti-CD73, anti-CD90, anti-CD105, anti-CD146, anti-CD14, anti-CD34, anti-CD45, and anti-HLA-DR; BD Biosciences) were incubated with samples for 30 min in PBS containing 0.5% BSA at 4 °C. Mouse isotypic IgG was used as a control.
FACS analysis of hMSC-exosomes was performed using exosome capture beads based on anti-CD63 coupled antibody (Invitrogen Dynabeads Exosome Human CD63 Isolation/Detection Reagent) following the manufacturer’s recommendations. Specific FITC-conjugated exosomal markers (anti-CD9 and anti-CD81; BD Biosciences) were used as above.

Human monoclonal antibodies AR3A, AR4A, and AR5A against HCV structural proteins were produced as described previously [27 (link);33 (link)]. Antibodies against HCV receptors were anti-CD81 (BD Pharmingen Cat. JS81), anti-SR-BI C16-17 [67 (link);77 (link)] and polyclonal anti-LDLr (R&D Systems Cat. AF2148). Control antibodies for receptor blocking assays were the isotype antibody 553447 for CD81, the antibody D for SR-BI [77 (link)], and a goat IgG (R&D Systems Cat. AB-108C) for LDLr. Soluble CD81 protein was a kind gift from Steven Foung [73 (link)]. Adapted recombinants with the core-NS2 sequence from genotypes 1a (H77), 2a (J6), 3a (S52), 4a (ED43), 5a (SA13) and 6a (HK6a) and UTR´s as well NS3-NS5B region from genotype 2a (JFH1) with or without HVR1 were described previously [44 (link);54 (link);58 (link)–61 (link)]. H77 E1E2 expression plasmids, previously validated for functionality in HCVpp assays [67 (link)], were used for E1/E2 interaction studies. Plasmids with point mutations were generated by conventional cloning techniques (Fusion PCR and Quikchange). The HCV sequence of the final plasmid DNA preparations were confirmed by direct sequencing (Macrogen). Antibody against NS5A, 9E10 [61 (link)], was provided by Charles Rice.

Before sorting, cells were washed twice in 1× PBS buffer (DPBS without calcium chloride and magnesium chloride; Sigma Aldrich, D8537) and labelled with 7-AAD (BD Pharmingen, 51-68981E) for live/dead differentiation and FITC-conjugated antibody [anti-CD47 (BD Pharmingen, 556045) for A375 and anti-CD81 (BD Pharmingen, 551108) for Jurkat]. After washing off the unbound antibodies in 1× PBS, cells were resuspended in BD FACS Pre-Sort Buffer (BD, 563503). Single cell sorting in 8-tube PCR strips was done using a BD FACSJazz Cell Sorter. A375 cells were sorted in 7 μl 1× PBS buffer and Jurkat cells in 8 μl lysis solution [100 μl 10× Lysis buffer (Takara, 635013), 5 μl RNase Inhibitor (Takara, 635013) and 700 μl water]. Following sorting, tubes were sealed and subjected to a quick spin and immediately frozen on dry ice and finally stored at −80°C until use. All sorting experiments included negative controls (no cell in a well).