Oval cell isolation

Oval cells (CD24⁺/Ter119^-) were isolated from 6-8-week-old Rag^-/- (n=6) resected livers derived from CCl₄-induced mice. A liver lobe, was resected under sterile conditions from each mouse and then was dissolved through cell strainer (100μm pore size) with simultaneous washes with sterile PBS (Phosphate-Buffered Saline, Thermo Fisher Scientific Inc. Gibco, Massachusetts, USA). Cells were centrifuged at 1,200xrpm for 5min and incubated with RBC 1X buffer [155mM NH4Cl (Sigma-Aldrich, Missouri, USA), 10mM NaHCO₃ (Sigma-Aldrich, Missouri, USA), 0.1mM EDTA (Sigma-Aldrich, Missouri, USA)] for 10min at 4°C. The reaction was terminated by FBS in a ratio of 1/10 RBC buffer. Afterwards, cells suspended in PBS buffer (0.1% BSA και 2mΜ EDTA) were incubated with 25μl rat anti-mouse CD24-FITC antibody (55326, Βecton Dickinson Biosciences, USA,) for 30min in a rotary motion at 4°C and centrifuged at 850xg for 8min. A 30min incubation protocol of cells with Sheep anti-Rat IgG Dynabeads (Ιnvitrogen, Massachusetts, USA) in PBS buffer was then followed and oval cells were finally separated using magnet (Ιnvitrogen, Massachusetts, USA). The oval cell isolation was confirmed by FACs assay for CD24⁺/Ter119^-.14,16