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It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O 2  •- ) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O 2  •- in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O 2  •- ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O 2  •- ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca 2+ (intracellular messenger) on the NO/O 2  •- ratio was also investigated. Graphical Abstract Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates.

analytical and bioanalytical chemistry/analytical and bioanalytical chemistry

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation