Spheroids Formation and Oxygen Measurement

Spheroids formation was performed following Wang et al. [29 (link)]. In brief, cells were seeded in 35 mm diameter dishes high Bioinert (Ibidi, Germany) at 0.1 × 10⁶ cells/dish density in complete medium supplemented with 25% of Methocel (Sigma-Aldrich) stock solution (0.5 g in 500 ml of complete medium). Spheroids size (µm) was determined by measuring feret’s diameter using standard ImageJ software tools (ImageJ, National Institutes of Health). Intracellular oxygen level was measured using BTP (bis(2-(20-benzothienyl)-pyridinato-N,C30) iridium (acetylacetonate) (Sigma-Aldrich), a fluorescent dye quenched by molecular oxygen [30 (link)]. Spheroids were stained with 5 µM BTP in complete medium for 4 h. After this time, spheroids were carefully washed with phenol-red free medium. Images of at least ten spheroids for condition were acquired using a Nikon C1si confocal microscope (Nikon). Fluorescence intensity was analyzed using standard ImageJ software tools.