10 cm ultra low attachment dish

Primary mammospheres were formed by culturing 4–5 × 10⁴ cells/well in ultra-low-attachment six-well plates (Corning, Inc.) in MEM that contained aliquots of recombinant epidermal growth factor (rEGF), fibroblast growth factor [FGF]-Basic, B27, and Gentamycin/Pen-strep B (all from Thermo Fisher, Waltham, MA, USA). For the secondary mammosphere formation assay, cells from monolayer culture were cultured as primary mammospheres in a 10-cm ultra-low-attachment dish (Corning, Inc.) for 4 days, and then those mammospheres were collected and dispersed with 0.05% trypsin, seeded in six-well ultra-low-attachment plates (10,000 cells/well) in mammosphere media, and counted a week later after having been stained with MTT (0.4 mg/mL, Sigma-Aldrich) to improve visualization of spheres. The number of spheres exceeding 80 μm in diameter was counted with a GelCount device (Oxford Optronix, Oxford, UK). Spheroid-forming efficiency (SFE) was calculated as the total number of secondary mammospheres (with a diameter larger than 80 μm) divided by total number of cells plated in serial dilutions of cell numbers. To evaluate the spheroid average size, 100 cells were plated in each well, and images of spheroid size were obtained with a Nikon eclipse Ti camera (NY, USA).