2.7. NADPH Oxidase Assay

NADPH oxidase activity was measured using a lucigenin-enhanced chemiluminescence method as described previously [28] with our own modification. Measurements of the increase in the kinetics of the luminescence were performed every 30 s for 15 min at 28 °C using an Infinite 200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The reaction was initiated by addition of 20 µL of cell extract (2 mg protein per ml) to the assay buffer (50 mM phosphate buffer with pH 7.0, containing 1 mM EGTA, 150 mM sucrose, 1 mM CaCl₂, 5 µM lucigenin and 200 µM NADPH). There was no measurable activity in the absence of NADPH. Activity was expressed as a relative rate of luminescence increase.