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 O2to generate O2.-was also observed directly similarly, occurring in milliseconds, with a rate constant k ∼ 3 × 106M-1s-1. Quinones act as redox mediators in LC.-/O2redox cycling. Oxidation of DCFH2to fluorescent DCF is not achieved by O2.-or H2O2, but NO2.reacts rapidly: k ∼ 1 × 107M-1s-1. Oxidation by H2O2requires 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.