Pitfalls in the Use of Common Luminescent Probes for Oxidative and Nitrosative Stress
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- Wardman, P., Burkitt, M.J., Patel, K.B. et al. Journal of Fluorescence (2002) 12: 65. doi:10.1023/A:1015363220266
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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.