Spheroid Viability Assay with Curcumin and PD98059

Spheroid culture was performed in an ultralow attachment 96-well plates as previously described [13 (link)]. Briefly, plates seeded with 10⁴ cells/well were centrifuged at 1,000 rpm for 10 min to allow cells to cluster in the wells and maintained in complete DMEM containing lactic acid (final concentration: 3.8 μM) for 5 days. Spheroids were treated with curcumin (40 μM) and/or PD98059 (50 μM) for 48 h. A two-color fluorescence assay was used to identify live and dead cells. Cell-permeable fluorescein diacetate (FDA, Sigma-Aldrich, 5 μg/mL) is converted into green fluorescent by esterases within living cells, whereas PI (Sigma-Aldrich, 10 μg/mL) enters the nucleus of dead or dying cells and emits red fluorescence upon binding to DNA. Phase-contrast images were acquired with a Leica inverted microscope. Spheroids were visualized using a Leica EL6000 fluorescence microscope (Leica Microsystems GmbH). Spheroid viability was determined according to the instructions provided with the Enhanced Cell Viability Assay Kit (Young In Frontier). Briefly, 10 μL of Cellvia solution was added to each well, kept at room temperature for 1 h, and then mixed by shaking for 1 min. The amount of formazan formed in living cells was measured spectrophotometrically at 450 nm using a GloMax-Multi microplate multimode reader (Promega Corporation).