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NADPH oxidase activity assay

TN Thi Thinh Nguyen
TU Trong Thuan Ung
SL Shinan Li
SL Sen Lian
YX Yong Xia
SP Sun Young Park
YJ Young Do Jung
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NADPH oxidase activity was assayed with lucigenin-enhanced chemiluminescence49. Briefly, HCT116 cells were harvested by cell scrapers and dounce homogenized in NADPH lysis buffer (50 mM phosphate buffer, pH 7.0, 1 mM EGTA, 150 mM sucrose, and protease inhibitors). Cell lysates were then incubated with 5 mmol lucigenin (Sigma) and 0.1 mmol NADPH (Sigma) balanced with NADPH lysis buffer. Photon emission from the chromogenic substrate lucigenin as a function of acceptance of electron/O2− generated by the NADPH oxidase complex was measured every 2 min for 30 min in a Berthold luminometer. The enzyme activity was expressed as relative light units/µg protein in 1 min, and relative fold changes were used to indicate the activity changes.

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