Ultraglo

Example 3

Human hepatocarcinoma cells (Hep G2) were plated into wells of 96-well white-walled assay tissue culture plates. Plated cells were incubated overnight in a humidified tissue culture incubator at 37° C. with 5% CO₂. The following day the superoxide probe, Compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile), was added to the cells in culture medium at a final concentration of 12.5 uM. Subsequently, dimethoxynaphthoquinone (DMNQ; 50 μM) or antimycin A (10 μM) diluted in culture medium was added to the reaction wells and the assay plate(s) were incubated for 1 hour in a humidified tissue culture incubator at 37° C. with 5% C02. For luciferin detection, the luciferin-utilizing luciferase enzyme, UltraGlo (Promega Corporation), was prepared by mixing Luciferin Detection Reagent (Promega), Reconstitution Buffer (Promega), and d-Cysteine (Promega), and one volume was added to the reaction. The plate was mixed using an orbital plate shaker, and luminescence was measured using a GloMax® luminometer. Data are shown in FIG. 4. An increase in signal was detected when cells were treated with either dimethoxynaphthoquinone (DMNQ) or antimycin A, both of which are known to induce formation of superoxide.

Example 2

The superoxide probe, Compound 1 (6-(4-hydroxy-3,5-dimethoxyphenoxy)benzo[d]thiazole-2-carbonitrile), was diluted into PBS to 25 μM into wells of a 96-well white-walled assay plate. Subsequently, generators of reactive oxygen and nitrogen species were diluted in PBS and added to the assay plate. The plate mixed using an orbital plate shaker for 5 minutes followed by a 30-minute incubation at room temperature protected from light. For luciferin detection, the luciferin-utilizing luciferase enzyme, UltraGlo (Promega Corporation), was prepared by mixing Luciferin Detection Reagent (Promega), Reconstitution Buffer (Promega), and d-Cysteine (Promega), and one volume was added to the reaction. The plate was mixed using an orbital plate shaker, and luminescence was measured using a GloMax® luminometer. Data are shown in FIG. 3; the blank includes PBS only; HX=hypoxanthine; XO=xanthine oxidase; and SOD=superoxide dismutase. The data demonstrate the presence of a strong signal in the presence of HX and XO, which are known to react to generate superoxide. This signal decreased in the presence of superoxide dismutase, confirming that superoxide is the source of the signal. Little to no signal was detected in the presence of hydrogen peroxide, Rose Bengal (which generates singlet oxygen), sodium nitrite, or NONOate.