Pe conjugated anti cd133 1 antibody

Populations of CD133 (putative marker of GSC) and EGFR positive cells were evaluated on a Coulter EPICS cytometer (Beckman Coulter, Fullerton, CA) as described previously [46 (link)]. Each sample was labeled with phycoerythrin (PE)-conjugated anti-human EGFR (EGFR.1) antibody (BD Biosciences, San Jose, CA) or PE-conjugated anti-CD133/1 (AC133) antibody (Miltenyi Biotec, Auburn, CA) according to the manufacturers’ recommendations. For BrdU labeling of proliferating cells in vitro, BrdU was added to tumorspheres at a final concentration of 10 μM 3 hrs prior to fixation. BrdU uptake was detected with a FITC BrdU Flow kit (B44, BD Biosciences) on dissociated single cells and analyzed with/without PE-conjugated anti-CD133/1 antibody (Miltenyi Biotec) anti-phospho-p38 MAPK (3D7, Cell Signaling Technology) according to the manufacturer's recommendation. CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit (Molecular Probes, Eugene, OR) and Annexin V-FITC apoptosis detection kit (BD Biosciences) were used according to manufacturers’ protocol. Appropriate compensation and isotype controls were used. All experiments were performed in triplicate.

The human hepatoma Huh7 cell line was obtained from the Human Science Research Resources Bank (Tokyo, Japan). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 μg/ml penicillin, and 0.25 μg/ml streptomycin and maintained in a humidified 37 °C incubator with 5% CO₂. Cells were harvested with 0.5 mM trypsin/EDTA (Invitrogen) and subsequently incubated at 4 °C with a phycoerythrin (PE)-conjugated anti-CD133/1 antibody (Miltenyi Biotec, Auburn, CA). LCSCs were sorted from Huh7 cells using flow cytometry (MoFlo XDP: Beckman Coulter, Miami, FL, USA) with an antibody against CD133/1. Isotype-matched mouse IgG was used as a control.