Apc conjugated anti epcam

For the determination of LPC percentages within liver NPCs, NPCs were incubated for 30 min on ice with the following antibodies: APC-conjugated anti-EpCAM and FITC-conjugated anti-CD45 (eBioscience). For the determination of the subtypes of liver leukocytes, liver leukocytes were incubated for 30 min on ice with the following antibodies: APC/FITC-conjugated anti-Ly6G, PE-conjugated anti-CD11b and FITC/APC-conjugated anti-CD45; FITC/PE-conjugated anti-CD8, PE-conjugated anti-CD4 and APC-conjugated anti-CD45 (eBioscience, San Diego, CA, USA). For the characterization of LPCs cultured in vitro, LPCs were trypsinized and incubated for 30 min on ice with the following antibodies: APC-conjugated anti-EpCAM, PE-conjugated anti-CD44, APC-conjugated anti-CD49f or FITC-conjugated anti-CD45 (eBioscience). For the detection of ROS, liver leukocytes were incubated with 2-[6-(4′-amino)phenoxy-3H-xanthen-3-on9-yl]benzoic acid (APF; Invitrogen, Carlsbad, CA, USA) fluorescence as reported before [26 (link)]. FCM measurements were performed with Aria II (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

LPCs were isolated as described previously [24 (link)]. Briefly, liver tissues were removed from CDE diet-fed mice after in-situ perfusion using a two-step collagenase perfusion method and then were incubated with 1 mg/ml collagenase D (Roche) and 1 mg/ml pronase (Roche) at 37 °C for 30 min. Non-parenchymal cells (NPCs) were separated from hepatocytes by repeated low-speed centrifugation. Next, NPCs were collected and suspended in a discontinuous gradient of 20% and 50% Percoll (GE Healthcare, Pittsburgh, PA, USA) and centrifuged continuously at 1400 × g for 20 min to enrich LPCs. The enriched LPCs were labelled with APC-conjugated anti-EpCAM and FITC-conjugated anti-CD45 (eBioscience, San Diego, CA, USA) antibodies, and EpCAM⁺CD45⁻ cells were isolated by fluorescence-activated cell sorting. These LPCs were cultured in type I collagen-coated dishes (BD Biosciences, San Jose, CA, USA). The standard culture medium for LPCs was DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum, 1 μg/ml insulin (Sigma-Aldrich), 1 × 10^− 7 mol/L dexamethasone (Sigma-Aldrich), 1% penicillin/streptomycin (Invitrogen), 50 ng/ml hepatocyte growth factor (PeproTech, Rocky Hill, NJ, USA), 20 ng/ml epidermal growth factor (PeproTech), 20 ng/ml FGF (PeproTech) and 1× insulin–Transferrin–Selenium–Ethanolamine (Invitrogen).