Cells were dissociated with Accutase and then resuspended in PBS containing 3% BSA. The collected cell suspensions were stained with FITC-conjugated human Ep-CAM antibody (Milteny) and Alexa Fluor APC-conjugated antibody against human C-Kit (B56; BD Biosciences) for 30 min on ice. EpCAM+/C-Kit− cells were sorted using a MoFloTM fluorescence-activated cell sorter. Sorted cells were seeded on Matrigel pre-coated plates and maintained in expansion basal medium containing small-molecule cocktails.
To analyze the cell proliferation, the expanded HBs were dissociated and then resuspended in PBS containing 10% FBS. The collected cell suspensions were fixed with 4% PFA (Sigma-Aldrich) and incubated in blocking and permeabilizing buffer, containing 0.1% Triton X-100, and 5% normal donkey serum in PBS for 30 min at room temperature. The cells were then incubated with APC-conjugated Ki67 (B56; BD Biosciences) and other indicated antibody for 30 min on ice. Corresponding isotype antibodies were used as controls. Flow cytometry analyses were performed using a FACS Aria II flow cytometer (BD Biosciences).
To analyze the cell proliferation, the expanded HBs were dissociated and then resuspended in PBS containing 10% FBS. The collected cell suspensions were fixed with 4% PFA (Sigma-Aldrich) and incubated in blocking and permeabilizing buffer, containing 0.1% Triton X-100, and 5% normal donkey serum in PBS for 30 min at room temperature. The cells were then incubated with APC-conjugated Ki67 (B56; BD Biosciences) and other indicated antibody for 30 min on ice. Corresponding isotype antibodies were used as controls. Flow cytometry analyses were performed using a FACS Aria II flow cytometer (BD Biosciences).