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Medium containing 3% agar, 10% FBS, 2 mM L-glutamine, and 5 μg/mL gentamicin was mixed with 8,000 cells/well suspension and then put into a 6-well plate to evaluate anchorage-independent cell growth. The indicated concentrations of DMSO and 3-DSC were then added. The plate was incubated at 37°C for 2 weeks, the colonies were pictured using a microscope (Leica Microsystems, Wetzlar, Germany), and the number of colonies was counted and evaluated.
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