Cells were collected and suspended in PBS, labeled with FITC mouse anti-human CD44 and PE mouse anti-human CD24 (BD Pharmingen TM), with 5 μl of antibody per one million cells in a final volume of 300 μl. A total of about 1.0× 106 cells were incubated with these two antibodies for 0.5 h at 4°C in the dark. Unbound antibody was washed off and the cells were analyzed on a BD FACS Calibur within 1 h of staining. Gating was established using the isotype controls FITC-labeled mouse IgG2a and PE-labeled mouse IgG2b (BD Pharmingen TM).
We used FACS to sort every passage of MCF-7 cells into two phenotypically distinct populations: CD44+/CD24- and all other phenotypes. The labeling method and condition were the same as described for the flow cytometric analysis.
We used FACS to sort every passage of MCF-7 cells into two phenotypically distinct populations: CD44+/CD24- and all other phenotypes. The labeling method and condition were the same as described for the flow cytometric analysis.