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Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" ,,,)

ted growth media with human gamma interferon (IFN-g) to induce cell differentiation. U-937 cells were assayed for superoxide anions after 2 to 5 days of differentiation with IFN-g. After 2 days of differentiation, superoxide anion activity was minimal (data not shown). However, after 3 days of differentiation, U-937 cells demonstrated superoxide anion activity upon stimulation with 100 ng/ml phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator and a potent stimulant of NADPH oxidase. Reaction samples were aliquoted into polystyrene round-bottomed tubes and placed into a luminometer to measure photon emission.

The luminometer was programmed to output raw data at 30-second counts. Detection of superoxide anion with luminol, enhancer, or PMA independently generated background numbers. Background values were determined against a control flask containing cells without differentiation.

Figure 2 shows the relative light units (RLUs) recorded by the luminometer for samples incubated at varying time points after 3 days of differentiation and control samples containing induced cells with luminol plus PMA, induced cells with luminol plus enhancer, induced cells with PMA plus enhancer, and induced cells alone. The results indicated that luminol oxidation by superoxide anion is time dependent. Superoxide anion activity was also assayed after 4 and 5 days of differentiation with IFN-g. The resulting RLUs yielded lower total RLUs and required a longer incubation period before substantial RLU values were established. The decrease in photon output coupled with the longer incubation period may be attributed to the health of the cultured cells.

Superoxide Generating System: Xanthine-Xanthine Oxidase

To demonstrate that superoxide anions interact with and oxidize luminol, we assayed a known superoxide anion generating system compris
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