Reactions were repeated a minimum of three times. Mouse tissues were lysed in 2× total protein buffer containing 10 mM of Tris (pH 7.6),
1% NP40, protease inhibitors (Roche complete mini ethylene diamine tetraacetic acid [EDTA]-Free), and 2 mM of dithiothreitol. Then, cell lysates were sonicated and quantified using the Bio-Rad Protein Assay. Then, 40 μg of protein were loaded into each well and separated by 4%-15% sodium dodecyl sulfate Mini-Protean TGX gel (Bio-Rad). After transfer, the polyvinylidene fluoride membrane was blocked with 5% nonfat dried milk, then cut into two pieces. The upper panel was click here incubated with rabbit anti-β-galactosidase (CEDARLANE); the lower panel was incubated with mouse monoclonal anti-β-actin (Sigma-Aldrich) at 4°C overnight. After washing, the peroxidase-conjugated secondary antibodies were added for 1 hour at room temperature. Detection was achieved using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL). Hepatocytes obtained from Sprague-Dawley GSK458 nmr rats were stained with PKH2 for cytoplasmic labeling, according to the manufacturer’s instructions (Sigma-Aldrich). After staining, hepatocytes were plated
on 24-well plates or T-150 culture flasks at a density of 1 x 104 cells/cm2. Fluorescence images of cells were obtained at predetermined time points on a Leica DMIL fluorescence microscope, using Leica application suite V3.1 software (Leica Microsystems, Buffalo Grove, IL). On days 1 and 14, the cells on flasks were collected and subjected to flow cytometry for quantitative analysis Demeclocycline of their fluorescence. For flow cytometric analysis, unlabeled cells were used as negative control. Mean fluorescent intensity was
determined for each sample, and total fluorescence was calculated by multiplying mean fluorescent intensity and the total number of cells. The value of total fluorescence on day 1 was given an arbitrary unit of 1. Total fluorescence on day 14 was calculated in an identical manner, then compared with that of day 1. Triplicates were used for statistical calculations. For flow cytometric analysis of hepatocyte purity, cells were fixed and permeabilized by 0.2% Triton X-100. After blocking with donkey IgG, cells were incubated with an anti-albumin/FITC (fluorescein isothiocyanate) antibody (1:20; CEDARLANE) for 30 minutes at room temperature. The negative control was FITC/rabbbit IgG. Fluorescence-activated cell-sorting acquisition was performed, using a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using FlowJo 6.4. A statistical analysis was done by the Student’s t test to identify significant differences. A P value less than 0.05 was considered significant. Flow cytometric analysis of LDPC purity was performed in an identical fashion, except for the antibody, which was PE-conjugated rabbit anti-CD45 antibody (BD Biosciences).