96 well ultra low attachment culture plates

Primary cells were suspended and seeded in6-well (coated with 1.5% agarose gel) or 96-well ultra low attachment culture plates(Corning). Cells were cultured with mesenchymal stem cell medium (Lonza, cat#190632 or cat#PT-3001), and incubated at 37°C in the humidified incubator with 5% CO₂ for at least 48 hrs for formation of spheroids. The spheroids were evaluated with the inverted microscope. Spheroid cultureswere maintained for up to 5–7 days and further processed for β-galactosidase staining.

Cells were seeded in 96-well ultra-low attachment culture plates (Corning, Tweksbury, MA) at 100 cells per well. Medium consisted of serum free DMEM/F12 Glutamax supplement medium (#10565042, Thermo Fisher Scientific), basis fibroblast growth factor (bFGF: 20 ng/ml, #13256029, Thermo Fisher Scientific), epithelial growth factor (EGF: 20 ng/ml, #PHG0313, Thermo Fisher Scientific), B-27 (1:50 dilution, #17504044, Thermo Fisher Scientific), and N2 supplement (1:100 dilution, #17502–048, Thermo Fisher Scientific). Ten days after seeding, photographs were obtained, and the sizes and numbers of sphere colonies on each well were counted using a microscope. Each experiment was repeated three times in triplicate.