Nrf2 activity assays Sample Clauses

Nrf2 activity assays. The HaCaT-ARE-Luc cell line contains the Nqo1 Antioxidant Response Element (ARE)-Luc reporter plasmid. The ARE is activated by all members of the CNC family of factors (Nrf1, Xxx0, Xxx0 and p45 NF-E2). The cells are cultivated in 24 well plates at the concentration of 2x104 cells/well in a CO2 incubator at 37ºC. For induction of Nrf2 activation the cells will be treated with increasing concentrations of the test substances for 6 h. As a positive control the cells will be treated with 0.2 mM Tert-butyl-hydroquinone (TBHQ). For inhibition of Nrf2, the cells are preincubated with increasing concentrations of test substances for 15 min and then treated with 0.2 mM TBHQ for 6 h. Then the cells are will behed twice in PBS and lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 7% glycerol during 15 min at RT in a horizontal shaker. After centrifugation, the supernatant are used to measure luciferase activity using an Autolumat LB 9510 (Berthold) following the instructions of the luciferase assay kit (Promega, Madison, WI, USA). The protein concentration in the cell extracts is measured by the Bradford method (BioRad). The RLU/mg is calculated, and the results are expressed as percentage of induction/inhibition of ARE-luc activity.
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