(Sigma, Lot#040M1344) to the drinking water at a concentration of 0.5 mg/ml coinciding with 1-2 weeks of DDC treatment. Total RNA was extracted from FACS sorted cells using the RNeasy Quantitative PCR. Mini Kit (Qiagen) and 30-50 ng was used to synthesize cDNA using the iScript cDNA Synthesis Kit (BioRad, 170-8890). Quantitative PCR was performed with SsoAdvanced™ SYBR® Green Supermix (Bio-Rad), 312 nM of Primer Mix (IDT) and cDNA template diluted 10-fold. Fold enrichment was determined using the difference of Ct method with values normalized to GAPDH. Primer sequences are listed in Supp. Table 2. Liver cell preparation. Livers of normal and DDC-treated mice were perfused initially with 1X HBBS solution followed by perfusion media (1X HBSS, 0.1 M CaCl2) containing 5mg/ml Liberase (Fisher Scientific). The digested liver and cells were further agitated by pipetting and collected through a 70-µm cell strainer for FACS analysis/collection. Flow cytometry staining. Cells were counted and blocked, followed by staining with EpCAM and IgG isotype control antibodies. After washing, cells were passed through a cell strainer and analysed/collected by FACSAria (Becton Dickinson) using DAPI to exclude dead cells. Immunostaining and quantification. Antibody staining was performed as previously described (Zong et al. 2009). Primary antibody sources and concentrations are listed in Supp. Table 1. The percentage of marker-positive cells, BrdU-labeled cells, and binucleated biliary cells was determined by taking representative images and directly counting cell number. For all quantitation, samples were obtained from at least three animals; cell enumerations for each experiment are listed in the text or Fig. legends.
(Sigma, Lot#040M1344) to the drinking water at a concentration of 0.5 mg/ml coinciding with 1-2 weeks of DDC treatment. Quantitative PCR. Total RNA was extracted from FACS sorted cells using the RNeasy Mini Kit (Qiagen) and 30-50 ng was used to synthesize cDNA using the iScript cDNA Synthesis Kit (BioRad, 170-8890). Quantitative PCR was performed with SsoAdvanced™ SYBR® Green Supermix (Bio-Rad), 312 nM of Primer Mix (IDT) and cDNA template diluted 10-fold. Fold enrichment was determined using the difference of Ct method with values normalized to GAPDH. Primer sequences are listed in Supp. Table 2. Liver cell preparation. Livers of normal and DDC-treated mice were perfused initially with 1X HBBS solution followed by perfusion media (1X HBSS, 0.1 M CaCl2) containing 5mg/ml Liberase (Fisher Scientific). The digested liver and cells were further agitated by pipetting and collected through a 70-µm cell strainer for FACS analysis/collection. Flow cytometry staining. Cells were counted and blocked, followed by staining with EpCAM and IgG isotype control antibodies. After washing, cells were passed through a cell strainer and analysed/collected by FACSAria (Becton Dickinson) using DAPI to exclude dead cells. Immunostaining and quantification. Antibody staining was performed as previously described (Zong et al. 2009). Primary antibody sources and concentrations are listed in Supp. Table 1. The percentage of marker-positive cells, BrdU-labeled cells, and binucleated biliary cells was determined by taking representative images and directly counting cell number. For all quantitation, samples were obtained from at least three animals; cell enumerations for each experiment are listed in the text or Fig. legends.
Supplemental_Materials.docx