Pitfalls in the Use of Common Luminescent Probes for Oxidative and Nitrosative Stress

Lucigenin (LC²⁺, bis-N-methylacridinium) and 2,7-dichlorofluorescin (DCFH₂) are widely used as chemiluminescent or fluorescent probes for cellular oxidative stress, to reflect levels of superoxide (O₂^·–) and hydrogen peroxide, respectively. We report mechanistic studies that add to the growing evidence for the unsuitability of either probe except in very well-defined circumstances. The ability for lucigenin to generate superoxide via reduction of LC²⁺ to LC^·+ and redox cycling with oxygen depends on the reduction potential of the LC²⁺/LC^·+ couple. Redox equilibrium between LC^·+ and the redox indicator benzyl viologen is established in microseconds after generation of the radicals by pulse radiolysis and indicated E(LC²⁺/LC^·+) –0.28 V vs. NHE. Reaction of LC^·+ with O₂ to generate O₂^·– was also observed directly similarly, occurring in milliseconds, with a rate constant k 3 × 10⁶M^–1 s^–1. Quinones act as redox mediators in LC^·+/O₂ redox cycling. Oxidation of DCFH₂ to fluorescent DCF is not achieved by O₂^·– or H₂O₂, but NO₂^·) reacts rapidly: k 1 × 10⁷M^–1 s^–1. Oxidation by H₂O₂ requires a catalyst: cytochrome c (released into the cytosol in apoptosis) is very effective (even 10 nM). Fluorescence reflects catalyst level as much as O₂^·–) production.