Figure 4.

Live and dead fluorescent assay for effect of continuous nonlethal hypoxia on PQ-induced cytotoxicity. Cells were exposed to normoxia (control), 8 h of treatment with 500 µM PQ + 24 h of normoxia (21% oxygen), and 8 h of 500 µM PQ + 24 h of hypoxia (1% oxygen). Culture dishes were subsequently stained with the live and dead assay (Molecular Probes) and viewed under a fluorescence microscope. A, B, and C show the Control, 8-h 500 µM PQ + 24-h normoxia, and 8-h 500 µM PQ + 24-h hypoxia cultures, respectively, with intracellular calcein fluorescence signifying viable cells. A', B', and C' show ethidium homodimer fluorescence of the same fields as A, B, and C, respectively, and demonstrate injured and nonviable cells. Note that PQ increased cell injury/death after 24 h, and this was diminished by nonlethal continuous hypoxia.