A total of 20,000 cells/cm2 were seeded to determine the cell viability by using the trypan blue vital cell dye (Sigma-Aldrich, St Louis, MO, USA) over 7 days.
Anchorage-independent growth was determined in 0.33% agarose (SeaPlaque, FMC BioProducts, Rockland, ME, USA) with a 0.5% agarose underlay. Cell suspensions (3300 cells) were plated in 60-mm diameter petri dishes in a semisolid medium IMDM 10% FBS. Dishes were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Colonies were counted after 7 days.
Anchorage-independent growth was determined in 0.33% agarose (SeaPlaque, FMC BioProducts, Rockland, ME, USA) with a 0.5% agarose underlay. Cell suspensions (3300 cells) were plated in 60-mm diameter petri dishes in a semisolid medium IMDM 10% FBS. Dishes were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Colonies were counted after 7 days.