Apc conjugated cd133 antibody

APC‐conjugated CD133 antibody was purchased from Miltenyi (cat. no. 130‐113‐184). PE‐conjugated CD24 antibody was obtained from BD Biosciences (cat. no. 555428). FITC‐conjugated CD326 antibody was purchased from STEMCELL (cat. no. 60136FI). Flow cytometry was performed according to the manufacturer's instructions.

After HCC cells were digested with trypsin, single-cell suspensions were stained with PE-conjugated CD13 antibody (BD Biosciences, Bedford, MA, USA, Cat. No. 555394) or APC-conjugated CD133 antibody (Miltenyi Biotech, Bergisch Gladbach, Germany, Cat. No. 130-113-668) in PBS containing 0.1% sodium azide and 2% FBS for 30 min on ice in the dark. Samples were washed once in staining buffer. Then, the cells were fixed with 1% (w/v) paraformaldehyde in PBS and preserved at 4 ºC. An isotype antibody was included as negative control. For apoptosis analysis, cells were stained with anti-CD13-PE and Annexin V-FITC (BioLegend, San Diego, CA, USA, Cat. No. 640906) or Annexin V-APC (BioLegend, Cat. No. 640920) resuspended in 300 μL binding buffer containing calcium ion for 30 min on ice in the dark. Samples were washed once in binding buffer. Apoptosis was immediately assessed by flow-cytometric analysis of Annexin-V staining in CD13+ cells. Flow cytometry was performed on a FACSCalibur machine (BD Biosciences).