CCK-8 cell proliferation assay

The MCF-7 cells and MDA-MB-231 cells were digested by 0.25% trypsin when they reached over 90% confluence, then centrifuged at 1,000 rpm for 5 min and re-suspended the cells. The cell concentrations were then adjusted to 5×10⁴/mL, and 100 μL of the cell suspensions were added to the 96-well plates (each well with 5,000 cells). The A-CM or U-CM were then added 6 h later to replace the previous medium. After 12, 24, or 48 h incubation, the co-culture medium was removed and replaced with 100 μL fresh medium containing 10 μL CCK-8, respectively, then incubated for 2 h. Subsequently, the optical density (OD) value of each well were observed and analyzed by a scanning microplate spectrophotometer at 450 nm (A450). The experiment was performed three times.