Cryopreserved sections (or fresh samples of tissue) were defrosted at room temperature and digested for 30 min at 37°C in a solution of Collagenase (2 ug/ml, Gibco)-Dispase (1 ug/ml, Gibco)-Bovine Fetal Calf Serum (2% FCS, Gibco) in Advanced DMEMF12 (LifeTech) supplemented with 1% Penicillin/Streptomycin (P/S, Gibco) and 1% L-Glutamine (Glut, Gibco). When the intrahepatic bile ducts could be observed by microscope inspection the sample was centrifuged at 100g for 5 mins, resuspended in Versene (Thermo Fisher) and incubated at 37°C for 1 hour. Dissociated cells were filtered through a 40 um cell strainer and washed in Advanced DMEMF12. Cells were resuspended in Advanced DMEMF12 underlayered with an equal volume of 20% and 50% (v/v) Percoll (Sigma). Following centrifugation at 1800g for 30 mins at 4°C, the HPC rich fraction lying between the 20 and the 50% Percoll layers was collected, washed and re-suspended in PBS with 2% FCS and 100 mmol EDTA (Sigma) for FACS staining procedure. Cells were incubated for 1 hour with EpCAM-BV650 conjugated antibody, CD24-BV421, CD133-APC, CD45-488 and CD31-488. Samples were stained with SYTOX-AADvanced dead cell stain kit (ThermoFisher) prior analysis in a FACSAria Fusion (BD Biosciences). Cells were grown as two-dimensional cultures and maintained as a cell line.
Label
SYBR Green
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
n/a
Description
12hrs hypoxia HL26 12hrs
Data processing
The normalization and data analysis were performed according to the manufacturers instructions using R Normalization uses the average expression of the housekeeping genes B2M, GAPDH and RPLP0. Housekeeping genes were chosen by the RT2 Profiler PCR Array software (GeneGlobe; Qiagen) Target gene signals were normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)]
