Igg1 pe

All cell samples were analyzed with a BD FACSCanto™ flow cytometry system (BD Bioscience, CA, USA), and statistical analysis was conducted in FlowJo software (FlowJo, OR, USA). The phenotype of T cells was assessed with fluorescently labeled antibodies specific for human CD3-PC5, CD4-fluorescein isothiocyanate (FITC) and CD8-FITC, which were obtained from BD Bioscience. Tumor surface antigen expression was detected with antibodies against human CD133-phycoerythrin (PE) (BioLegend, CA, USA) and GPC3-PE (Abcam, MA, USA); isotype control groups were stained with IgG1-PE (Abcam). The expression of GFP in T cells was evaluated FL1 channel to demonstrate the expression of CD133-CAR. The expression of GPC3-CAR was assessed by recombinant biotinylated protein L (Thermo Fisher Scientific) binding PE-conjugated streptavidin (PE-SA, BD Bioscience). All FACS-related cell samples were handled on ice and washed three times with 1 × PBS (Thermo Fisher Scientific) containing 1% FBS before staining the corresponding antibodies.

MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, USA) at 37°C in an atmosphere of 5% CO₂. The stem-like ALDH^hiCD44⁺ cells and non-stem-like ALDH^lowCD44⁺ cells were separated from the MDA-MB-231 cell culture using fluorescence-activated cell sorting with an ALDEFLUOR™ assay kit (Stemcell Technologies, Inc., Vancouver, BC, Canada), and monoclonal fluorescence-conjugated rat anti-mouse antibodies against CD44-phycoerythrin (PE; 1:100) and immunoglobulin (Ig) G1-PE (1:100; Abcam, Cambridge, UK), according to the manufacturer’s instructions. Briefly, the cells were incubated in ALDEFLUOR assay buffer containing ALDH substrate. In each experiment, a sample of cells was incubated with 50 mmol/l diethylaminobenzaldehyde (a specific ALDH inhibitor) at 37°C for 45 min, for use as the negative control. Subsequently, the CD44 and IgG1 antibodies were added with ALDH into negative control Eppendorf tubes at a temperature of 4°C for 30 min. Finally, all the cells were resuspended with buffer for detection by flow cytometry.