Stemxvivo serum free media

Single cell was plated in ultralow attachment 6-well plates (Corning) at 25,000 cells per well for primary tumorsphere formation. After incubation for 7 days, mammospheres were collected and dissociated by trypsin. As the secondary tumorsphere formation, 200 cells per well were plated in ultralow attachment 96-well plates. Cells were grown in StemXVivo Serum-Free Media (R&D) containing 2 U/ml Heparin (Tocris) and 0.8 μg/ml Hydrocortisone (Tocris). Mammospheres were harvested 7 days later, and tumorsphere was calculated under inverted phase-contrast light microscope (Olympus). The experiment was repeated three times. Statistical difference on secondary tumorsphere formation was assessed using student T-test with p ≤ 0.05 as the significance criterion.
When the effect of IL6 on tumorsphere formation was examined, recombinant human IL6 (Peprotech) was supplemented at concentration of 0, 100, 200 ng/μl in the beginning of the primary and secondary tumorsphere formation stages.

Caki-2 or A498 cells were seeded on ultralow attachment 6-well plates (Corning, NY, USA) at 5×10³ cells per well for primary tumorsphere formation. After incubation for 2 weeks, tumorspheres were collected and enzymatically dissociated by trypsin. For secondary tumorsphere formation, 2000 cells per well were plated in ultralow attachment 96-well plates again. Cells were grown in StemXVivo Serum-Free Media (R&D systems) supplemented with 2 U/ml heparin (H8060, Solarbio, Beijing) and 0.8 μg/ml hydrocortisone (G8450, Solarbio, Beijing). Two weeks later, tumorspheres were observed and analyzed under an inverted phase-contrast light microscope (Olympus).

Cells were plated in 6-well plates at a density of 4×10⁵ cells/well in 2 mL of complete culture medium, and incubated with PAM for 24 h, harvested and dosed. 200 cells were plated in 200 μL StemXVivo Serum-Free Media (R&D) containing 2 U/mL heparin (Tocris), 0.8 μg/mL hydrocortisone (Tocris), 100 U/mL penicillin (Solarbio, Catalog# P1400), and 100 μg/mL streptomycin (Solarbio, Catalog# P1400) in ultra-low adherent 96-well plates (Corning). Colonies were quantified after 7 days.