Ultra low attachment surface 24 well plates

Cells were seeded into 24-well ultra low-attachment surface plates (Corning) at a density of 1500 cells/well and were cultured in serum-free RPMI1640 supplemented with 1.5% B27 (Life technologies), 20 ng/ml mouse recombinant EGF and 10 ng/ml FGF (Peprotech). Conditioned medium was prepared identically, but without adding EGF and FGF. Spheroids formed within 5 to 6 days after seeding.

Mouse ascites with tumoroids were collected. Tumoroids were washed with RBC lysis buffer 1-2 times, then plated in 24-well ultra-low attachment surface plates (#33019010, Corning). Tumoroids were cultured in a 1:1 mixture of OV90 medium and advanced DMEM/F12 medium (#2322978; Thermo Fisher Scientific) containing 4% B27 (#A1895601, Life Technologies), 2% N-2 supplement (#11520536, Thermo Fisher Scientific) and 0.04% EGF (#PMG8045, Invitrogen).

The spheroid-formation ability of BCSCs were determined using tumour sphere formation assays and single-cell tumour sphere formation assays, respectively. For the tumour sphere formation assays, BCSCs were collected after transfection with the corresponding vector for 48 h; then, 1 × 10² BCSCs were seeded on an ultra-low attachment surface 24-well plates (Corning, USA). BCSCs were resuspended in DMEM/F-12 supplemented with 20 ng/mL EGF, 20 ng/mL bFGF and 2% B27 and incubated for 7 days at 37 °C. Finally, the spheres were visualized under an optical microscope (Olympus, Japan). For the single-cell tumour sphere formation assays, BCSCs were collected after transfection with the corresponding vector for 48 h; then, one BCSCs were seeded on an ultra-low attachment surface 96-well plates (Corning, USA). BCSCs were resuspended in DMEM/F-12 supplemented with 20 ng/mL EGF, 20 ng/mL bFGF and 2% B27 and incubated for 7 days at 37 °C. Finally, the spheres were visualized under a confocal laser-scanning microscope (Leica, Germany).