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Histological analysis of hypoxia revealed no significant differences between spheroids of different diameters. Pimonidazole (green) and DAPI (blue) staining further verified the absence of a hypoxic core in the 15 000-, 30 000- and 60 000-cell spheroid. A 60 000-cell spheroid was incubated in 1% oxygen for 5 days as a positive control for pimonidazole staining (right). Scale bar represents 500 µm; images were captured using a 20× objective.
