A1 scanning confocal system

Oxygen uptake rate distribution was determined using a hypoxia kit, Image-IT™ Green Hypoxia Reagent (Thermo Fisher Scientific, cat. no I14834). To assess the oxygen uptake rate distribution in the spheroids, WM266-4 and WM115 cells were seeded at densities of 1000 and 2000 cells/drop. On days 4 and 8, the spheroids were transferred to glass-bottom dishes, and 20 μl of Image-IT™ Hypoxia Reagent (10 μM) was added to each spheroid. The spheroids were incubated at 37 °C for 1 h. The hypoxia dye was exchanged with fresh growth medium, and the spheroids were placed again in a cell culture incubator for the next 4 h. Spheroid images were taken with a Nikon Eclipse Ti-E microscope coupled to an A1 scanning confocal system (Nikon, Japan) at a 488 nm/520 nm fluorescent wavelength.

To evaluate glucose uptake, a fluorescence-emitting d-glucose probe 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (Thermo Fisher Scientific, cat. no N13195) was used. To assess the oxygen uptake rate distribution in the spheroids, WM266-4 and WM115 cells were seeded at densities of 1000 and 2000 cells/drop. On days 4 and 8, the spheroids were transferred to glass-bottom dishes, and 20 μl of 2-NBDG (200 μM) was added to each spheroid. The spheroids were incubated at 37 °C for 45 min. Finally, spheroids were washed with PBS, and spheroid images were taken with a Nikon Eclipse Ti-E microscope coupled to an A1 scanning confocal system (Nikon, Japan) at a 494 nm/551 nm fluorescent wavelength.