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Published in final edited form as: Free Radic Biol Med. 2007 Dec 8;44(5):868–881. doi: 10.1016/j.freeradbiomed.2007.11.020

Fig. 5 — (A) K562/NOX5 cells were loaded with fluo-4 (green fluorescence, calcium influx) and HE (red fluorescence, superoxide production), then stimulated with 100 μM H₂O₂. The effects of BAPTA (50 μM), DPI (50 μM), or SOD (400 U/ml) on NOX5 regulation by H₂O₂ were analyzed. Confocal images using a Zeiss microscope were recorded for up to 30 minutes using dual excitation 488/543nm. Images from the 0 and 5 minute time points are shown. Each set of four photomicrograph panels correspond to calcium influx (green fluorescence – top left), superoxide production (red fluorescence - top right),, the differential interference contrast (DIC) image (bottom left), and a merge picture of the fluorescent images (bottom right). The scale bar indicates a size of 10μm. (B) The fluorescence intensity levels from a representative experiment are shown using either K562/NOX5 cells (black line) or K562 cells treated with H₂O₂ (blue line). Fluorescence levels in K562/NOX5 cells in the absence of H₂O₂ were also analyzed (brown line). After normalization by subtraction of the zero-time value, fluorescence was plotted as the mean for a field of approximately 15 cells. For each data set, curve-fitting analysis was carried using Excel and the best curve fit shown together with the coefficient of correlation. (C) HEK/NOX5 cells were loaded with fluo-4 and HE and confocal imaging carried out as in (A) using a single excitation wavelength (488nm).

Fig. 5 — (A) K562/NOX5 cells were loaded with fluo-4 (green fluorescence, calcium influx) and HE (red fluorescence, superoxide production), then stimulated with 100 μM H₂O₂. The effects of BAPTA (50 μM), DPI (50 μM), or SOD (400 U/ml) on NOX5 regulation by H₂O₂ were analyzed. Confocal images using a Zeiss microscope were recorded for up to 30 minutes using dual excitation 488/543nm. Images from the 0 and 5 minute time points are shown. Each set of four photomicrograph panels correspond to calcium influx (green fluorescence – top left), superoxide production (red fluorescence - top right),, the differential interference contrast (DIC) image (bottom left), and a merge picture of the fluorescent images (bottom right). The scale bar indicates a size of 10μm. (B) The fluorescence intensity levels from a representative experiment are shown using either K562/NOX5 cells (black line) or K562 cells treated with H₂O₂ (blue line). Fluorescence levels in K562/NOX5 cells in the absence of H₂O₂ were also analyzed (brown line). After normalization by subtraction of the zero-time value, fluorescence was plotted as the mean for a field of approximately 15 cells. For each data set, curve-fitting analysis was carried using Excel and the best curve fit shown together with the coefficient of correlation. (C) HEK/NOX5 cells were loaded with fluo-4 and HE and confocal imaging carried out as in (A) using a single excitation wavelength (488nm).

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