Summit version 4

After 5 days of culture, mouse fetal HpSCs were harvested with 0.05% trypsin (Sigma). Cells were washed with phosphate-buffered saline and cellular apoptosis was detected by Annexin-V FLUOS staining kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions. Samples were stained with 10 μg/mL propidium iodide in the dark at 4°C for 10 minutes. Annexin V-positive cells were analyzed with MoFlo and the Summit version 4.0 software (both DakoCytomation).

Mouse hepatic stem cells were sorted from the liver of embryonic day 13.5 C57BL/6 fetal mice (n = 4), as we described previously [31 (link)]. In brief, liver cells were stained with the following antibodies: biotinylated anti-Ter-119 and anti-CD45, anti-c-Kit-APC, anti-CD49f-PE, and anti-CD29-FITC. Streptavidin-labeled allophycocyanin-Cy7 was used to detect biotinylated antibodies. All the above antibodies were purchased from BD Pharmingen, San Diego, CA, USA. For gating, the CD45⁻Ter119⁻ c-Kit⁻ cell population was firstly gated out, and then the CD49f^+/lowCD29⁺ subpopulation was set as the sorting gate. Analysis and sorting were performed using MoFlo with Summit version 4.0 software (DakoCytomation, Denmark).

Suspended liver cells were obtained from embryonic day 13.5 C57BL/6 fetal mice as previously described [33 (link)]. In brief, dissociated liver cells were stained with biotinylated anti-CD45 and Ter-119 monoclonal antibodies, anti-CD29-FITC antibody, anti-CD49f-PE antibody, and anti-c-Kit-APC antibody. Cells positive for the biotinylated antibodies were detected with streptavidin-labeled allophycocyanin-Cy7 (all monoclonal antibodies were purchased from BD PharMingen, San Diego, CA, USA). Labeled cells were then analyzed and separated using MoFlo (DakoCytomation, Denmark) and Summit version 4.0 software (DakoCytomation, Denmark).