Anti epcam antibody

After the gradient test, the mononuclear cells were incubated with 100 μl of micromagnetic beads coated with anti-EpCAM antibody (Miltenyi-Biotec) and 100 μl of FcR-blocking reagent (Miltenyi-Biotec) for 30 minutes at 4°C and then incubated with 20 μl of magnetic beads coated with anti-CD45 antibody (Miltenyi-Biotec) for 15 minutes at 4°C. The mixture was passed through a magnet-filled column with an AutoMACSPro cell separator system (Miltenyi-Biotec) using the positive selection protocol (POSSELD program) to enrich for EpCAM-positive cells and the negative selection protocol (DEPLETE program) to enrich for CD45-depleted CTCs. The CD45-depleted (i.e., CD45-negative) fraction underwent an additional run through the magnet-filled column using the MACS DEPLETES program to prevent any residual contamination of CD45-positive cells.

Mice were fed with 0.1% DDC (Sigma-Aldrich, St. Louis, MO) for 3 weeks to induce the activation of OCs. OCs were isolated as we previously reported [26 (link)]. Briefly, livers were perfused from portal vein with collagenase and pronase digestion. Liver cells were resuspended and centrifuged for collection of NPCs. NPCs were centrifuged through a discontinuous gradient of 20% and 50% Percoll^TM (Amersham Biosciences, Pittsburgh, PA). Finally, OCs were isolated from these cells fragment using a MiniMACS column containing anti-EpCAM antibody (Miltenyl Biotec, Auburn, CA). The EpCAM⁺ cells were tested for OC markers by flow cytometry and determined to contain OCs at approximately 95% purity. Cells were cultured on type I collagen coated dishes (BD Biosciences, San Jose, CA) in complete medium. Single clones were isolated and expanded in vitro.