Abcb1

One million Y79 cells were stained by incubating with directly labelled primary antibodies (CD133-Miltenyi Biotech, CD44, CD90, CXCR4-Ebioscience and ABCB1-Abcam) for 45 min at 4 °C. The antibodies were standardized by varying their dilutions and checking the expression percentage. The cells were then washed thrice with wash buffer to remove excess antibody and run in the BD LSRFortessa™ flow cytometry analyser and the analysis was done using FACSDiva™ software version 6.2. Appropriate controls were used for the experiments. A total of 20,000 to 50,000 events were acquired for analysis. The cells were gated based on size, granularity and doublet discrimination as described previously [18 (link)]. In brief, Y79 cells were first selected based on forward and side scatter, and the doublets were excluded using the doublet discrimination plots. The negative control (unstained cells) is used to set the laser voltages, sorting gates and establish the Allophycocyanin (APC) expression profile for the population. The labeled cells were then run through the cytometer and the two populations (CD133hi and lo) are collected in tubes with medium containing 2X antibiotic solution. The post-sort purity and viability was determined. The sorted cells were then used for CSC characterization.