Cellular hypoxia assessed by pimonidazole staining. Human osteosarcoma cells (U2OS) were grown in oxygen-impermeable dishes and incubated in the absence or in the presence of either antimycin A (1 μg/mL) or rotenone (1 μM) in hypoxia for 4 hours. Normoxic, untreated cells served as a control. Intracellular pimonidazole complexes, which are formed under hypoxic conditions (pO₂ less than 10 mm Hg), were detected by fluorescence microscopy using an FITC-labeled monoclonal antibody. Pimonidazole was applied to the cells for 45 minutes. Cell nuclei were visualized by bisbenzimide staining. Cells were exposed to (A) intermediate hypoxia (3% O₂) or (B) to almost anoxic conditions (0.1% O₂).