Assay Method Information

Assay Name:  Nox4 Assay
Description:  Cells: HEK (CJ Nox4) stably expressing Nox4 was purchased from Redoxis AB (Lund). The adherent cells were cultivated in RPMI 1640 with L-Glutamine were supplemented with FBS (10%), penicillin (10 U/ml) streptomycin (100 μg/ml) and neomycin (200 μg/ml) at 37 C. in air with 5% CO2.Hydrogen peroxide produced by Nox4 was measured (fluorescence emission: 590 and excitation: 544) using Amplex red (Molecular Probes) in Fluorescan Ascent plate reader Type 374. Cells were collected from cultures by Trypsin mediated detachment of adherent cells. Cells were seeded in 96-well plates at a density of 50 000 cells in 200 l assay volume. Inhibitors were added for 30 min (37 C.) and then reagents was added to give a final concentration of Amplex Red 35 mM and 0.17 U/ml horseradish peroxidase. Nox4 activity was measured up to 100 min with readings every minute. Inhibition curves of different Nox4 inhibitors were evaluated at 50% inhibition (IC50) in comparison to cell control without inhibitor present. Y-axes: turnover of hydrogen peroxide; x-axes: concentration of inhibitor. Inhibitors were diluted in a compound plate in DMSO (100%) then transferred to Hanks buffer solution and in assay plate DMSO were 2% in all the wells.Compounds (Nox inhibitors) were diluted at 3 working concentration and titrated from 200 μM to 0.003 μM in 11 steps.
Affinity data for this assay

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