Effects of illumination intensity on the photo-oxidation of Amplex red. (A) Three neutral density filters – Filter A (transmittance=22%), Filter B (7.6%) and Filter C (0.85%) – were used to attenuate the illumination intensity of the visible light source of the UV–Vis spectrophotometer. (B) The resorufin concentration was determined for a reaction mixture containing 280±6 μm AR, 5.5±0.1 nm HRP and 27±0.5 μm H2 O2 after 300 s in the dark (in gray) and after illumination every 0.1 s for 300 s. The illumination intensity was decreased with the use of neutral density filters. The experiments were repeated in triplicate. The data show that the illumination intensity does not significantly affect the final concentration of resorufin, which means that photo-oxidation of Amplex red is not occurring in the time scales and optical powers used in these experiments.