Isolation of Hepatic Oval Cells from DDC-treated Mice

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.