Epcam

iCCA samples were subjected to mechanical and enzymatic dissociation, as previously described in our laboratories^{29 (link),30 (link)}. In brief, the tissue was minced with surgical scalpels into fragments of approximately 1 mm³. After cutting, the tissue was washed with Dulbeccos’ PBS by bench centrifugation and re-suspended. After washing, fragments were transferred into digestion solution (growth medium with 1 mg/mL collagenase type IV, 0.1 mg/mL hyaluronidase, and 0.1 mg/mL DNase), and incubated for 12 to 16 h at 37 °C in a humidified atmosphere of 5% CO₂ in air. Then, the cell suspension was filtered with a 100-mm cell strainer placed on a 50-mL tube. The cell strainer was washed with 5 mL of growth medium. The cell suspension was then filtered with a 70-mm cell strainer placed on 50-mL tube. The cell strainer was washed with 5 mL of growth medium.
For magnetic cell sorting, cells were labeled with CD326 MicroBeads (EpCAM, Miltenyi, 130-061-101), and sorted using the Miltenyi Biotec Cell Isolation Kit, according to the manufacturer’s instruction. Isotype-matched mouse immunoglobulins served as controls. Finally, the cells were re-suspended in growth medium and placed into 6-well dish at 37 °C in a humidified atmosphere of 5% CO₂ in air.

the following antibodies were used at a concentration of 1 μg Ab/mL sample: EpCAM (Biolegend), CD151 (Miltenyi), and EGFR (Thermo Fisher). The isotype controls used were the Biolegend IgGk2b (to match EpCAM), Miltenyi REA Isotype control (to match CD151), and the Biolegend IgGK1 (to match EGFR). Notably, the liver cancer EV subpopulation isolation required filtering with a .45 μm filter unit (GE Whatman) to achieve strong specificity versus isotype control. Samples were run at a flow rate of = 2.5 mL/hr.

Huh-7 cells were electroporated with JFH1 RNA, a genotype 2 strain of HCV as previously described (70 (link)). Upon confirming >85% infection rate by HCV NS5A immunofluorescence, Huh-7 cells were plated in 48 well plates. Day 6 Mϕs were spun down onto Huh-7 cells at 400× g for 5 min at a ratio of 2:1. Following 24 h incubation, macrophages were removed by pipetting, and Huh-7 cells detached using Accutase (Sigma-Aldrich). Huh-7 cells were labeled with Annexin V (Becton Dickinson, 550474), Epcam (Miltenyi Biotec, 130-091253) and propidium iodide (Sigma Aldrich, P4864) in 1× Annexin V binding buffer according to the manufacturers protocol. Cells were washed thoroughly, fixed with 2% paraformaldehyde and analyzed on the BD Biosciences LSR II cell analyzer.