Anti human cd133 apc

To minimize experimental variability and loss of cell viability, all experiments were performed on fresh tumor cell suspensions prepared before flow cytometry. Antibody staining was performed in PBS 1× containing 1% bovine serum albumin and 2% fetal bovine serum. Analysis of ALDH enzymatic activity was performed using the Aldefluor system (StemCell Technologies Inc., France) according to the manufacturer’s instructions. The cells were subsequently washed and stained with antibodies. Antibodies used in this study included: anti-human CD44-Pacific Blue (Exbio, Clinisciences, France), anti-human CD133-APC (Miltenyi Biotec, France) and anti-human CD166-PE (Beckman Coulter, France). Flow cytometry analysis was performed by using a MoFlo Astrios cell sorter (Beckman Coulter, France).

Cultured cells were washed with phosphate-buffered saline (PBS) and resuspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) at a density of 1×10⁵ cells/100 µL. Single cells were stained using antibodies against CSCs; anti-human CD133-APC (Miltenyi Biotec, NRW, Germany), anti-human CD44-FITC (Miltenyi Biotec), and anti-human CD24-FITC (Miltenyi Biotec), and the Human MSC Analysis Kit (BD Biosciences, NJ, USA); CD90-FITC, CD105-PerCP-Cy5.5, and CD73-APC. Cells were incubated with the antibodies for 15 min at room temperature, washed with PBS, and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences) and the software FlowJo v.10.