Bioluminescent Imaging of ROS

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (bis-N-methylacridinium nitrate) are used to detect reactive oxygen species (ROS). Luminol bioluminescence detects mainly to neutrophil myeloperoxidase (MPO) activity, and lucigenin bioluminescence detects NADPH oxidase activity and extracellular superoxide production of macrophages (39 (link),40 (link)). Na-luminol and lucigenin (Santa Cruz Biotechnology) were dissolved in phosphate buffered saline to form 20-mg/ml and 2.5-mg/ml stock solutions, respectively. Anesthetized mice were injected intraperitoneally with 150 mg/kg luminol (days 0, 1, 2, and 4) or 25 mg/kg lucigenin (days 0, 2, 6, and 10). Bioluminescence imaging was performed 10 minutes postinjection using an IVIS Lumina II. Acquisition time was 60 seconds, F-stop 1, Binning 8. Data were analyzed and ROIs were applied; luminescence was expressed as total radiance (photons/second/cm²/steradian) within the ROIs.

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Botz B., Bölcskei K., Kereskai L., Kovács M., Németh T., Szigeti K., Horváth I., Máthé D., Kovács N., Hashimoto H., Reglődi D., Szolcsányi J., Pintér E., Mócsai A, & Helyes Z. (2014). Differential Regulatory Role of Pituitary Adenylate Cyclase–Activating Polypeptide in the Serum-Transfer Arthritis Model. Arthritis & Rheumatology (Hoboken, N.j.), 66(10), 2739-2750.

Publication 2014

lucigenin

Bis n methylacridinium Lucigenin Luminescence Luminol Macrophages Mice Myeloperoxidase Nadph oxidase Neutrophil Nitrate Phosphate Reactive oxygen species Saline Superoxide

Corresponding Organization : University of Pecs

Other organizations : Semmelweis University, Montavid Thermodynamic Research Group, Osaka University of Pharmaceutical Sciences

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Protocol cited in 9 other protocols

Variable analysis

independent variables

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) concentration (150 mg/kg)
Lucigenin (bis-N-methylacridinium nitrate) concentration (25 mg/kg)

dependent variables

Neutrophil myeloperoxidase (MPO) activity (detected by luminol bioluminescence)
NADPH oxidase activity and extracellular superoxide production of macrophages (detected by lucigenin bioluminescence)

control variables

Phosphate buffered saline (used to dissolve luminol and lucigenin)
Anesthetized mice
Bioluminescence imaging parameters (10 minutes post-injection, 60 seconds acquisition time, F-stop 1, Binning 8)

positive controls

None specified

negative controls

None specified

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