Pe conjugated cd133

HepG2 or MHCC-LM3 cells were first labeled with primary antibody for CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany) and then incubated with goat anti-mouse IgG microbeads (Miltenyi Biotec) according to the manufacturer’s protocols. Cells were then sorted magnetically using MACS LS columns (Miltenyi Biotec). After dead cells were excluded from the sort via an electronic gate, cells expressing CD133 were collected through a sort gate. In addition, phycoerythrin (PE)-conjugated CD133 (BD PharMingen, San Jose, CA, USA) were used in the experiment. The processed cells were incubated in phosphate-buffered saline (PBS) containing 2% FBS followed by PE-conjugated antibodies. Isotype-matched mouse immunoglobulins served as controls. The samples were analyzed using a FACSCanto II analyzer flow cytometer (BD Biosciences, San Jose, CA, USA).

FACS analysis and cell sorting were performed using a FACSCanto II and FACSAria II (Becton Dickinson, Franklin Lakes, NJ), respectively. FACS data were analyzed using FACS Diva software (BD). Antibodies to the following proteins were used: FITC-conjugated CD44 (MACS, Miltenyi Biotech Korea, Seoul, Korea) and PE-conjugated CD133 (BD). FACS gates were established by staining with isotype-matched PE or FITC conjugated antibodies (BD).

The HCC cell lines were washed in phosphate-buffered saline (PBS), which included 2% FBS. The suspension cells were incubated with fluorochrome-conjugated antibody at 4°C for 30 minutes. Flow cytometry (FACS) analysis was performed using the FACS Canto II system (Becton Dickinson, Franklin Lakes, NJ, USA). The FACS data were analyzed using FACS Diva software (Becton Dickinson (BD)). Antibodies to the following proteins were used: fluorescein isothiocyanate (FITC) or allophycocyanin (APC)-conjugated CD44 (MACS) and phosphatidylethanolamine (PE)-conjugated CD133 (BD). The FACS gates were established by staining with isotype antibody conjugated FITC, APC and/or PE (BD).