4.8. Sphere Formation Assay

HRTPT cells were cultured in a T-25 flask and allowed to reach confluency. Cells were refreshed with a medium supplemented with either 12.5 nM bortezomib or Dimethyl sulfoxide (DMSO) as a control for 48 h, after which cells were detached with the TrypLE enzyme and 1000 cells/cm² were seeded into a Corning™ T-25 ultra-low attachment flask (#07-200-876, Thermo Fisher Scientific) supplemented with 6 mL of DMEM: F12 serum-free medium with either 12.5 nM bortezomib or DMSO as a control. Cells were allowed to grow undisturbed for seven days at 37 °C in a 5% CO₂ incubator. The spheres were harvested from the flask and transferred to a 15 mL tube. The flask was rinsed with 2 mL of DMEM/F12 and combined with the collected suspension, followed by centrifugation at 200× g for 5 min. The supernatant was carefully aspirated, ensuring the sphere pellet was not disturbed, followed by gentle resuspension to a final volume of 500 μL with DMEM/F-12. Then, 50 μL of the spheres suspension was added to each well of the 96-well plate, and the spheres were visualized and counted in each well under a microscope. Sphere formation efficiency was calculated by dividing the number of spheres counted by the number of seeded cells then multiplying by 100.