The cells were detached with Accutase® solution (Sigma, St Louis, MO), washed, re-suspended in ice-cold PBS, and then incubated for 30 min at 4°C with the following anti-human fluorophore-labeled monoclonal antibodies: CD44-APC (clone BJ18, BioLegend, San Diego, CA), CD133-APC (clone AC133, Miltenyi Biotec Inc., San Diego, CA), CXCR4-APC (clone 12G5, BioLegend, San Diego, CA), and CD24-FITC (clone ML5, BioLegend, San Diego, CA). Cells (5x103/sample) were counted using a FACS (Beckman Coulter Gallios™, Indianapolis, IN). The data were analyzed with Kaluza analysis software. For specific binding analysis, cells (2x106) were incubated with CXCR4-KLA-FITC (10 μM) for 2 h at 37°C in 1.5% (w/v) FBS-containing medium. Subsequently, cells were detached, washed, re-suspended in ice-cold PBS, and FACS analysis was performed to determine the binding of a FITC-labeled CXCR4-KLA to CXCR4Low and CXCR4High. Cells (2x104/sample) were counted using a FACS (Beckman Coulter Gallios™, Indianapolis, IN) and the data were analyzed with Kaluza analysis software.
Cell sorting was performed using FACSVantage (BD Bioscience, CA). Cells (2x107) were stained with CXCR4-APC (clone 12G5, BioLegend, San Diego, CA) for 1 h at 4°C under sterile conditions. The isolated CXCR4Low and CXCR4High subpopulations were further characterized for purity by FACS analysis, as described above.
Cell sorting was performed using FACSVantage (BD Bioscience, CA). Cells (2x107) were stained with CXCR4-APC (clone 12G5, BioLegend, San Diego, CA) for 1 h at 4°C under sterile conditions. The isolated CXCR4Low and CXCR4High subpopulations were further characterized for purity by FACS analysis, as described above.