ROS levels in primary MEM cells cultured in different concentrations of glucose or treated with t-BOOH (Sigma-Aldrich) were determined after incubation for 30 minutes in the dark with the fluorescent probes 5-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA, 0.5 μM; Invitrogen) or dihydroethidium (DHE, 2 μM; Invitrogen). CM-H2DCFDA is a general ROS indicator that detects primarily hydrogen peroxide, whereas DHE specifically detects the superoxide anion66. Cell viability was assessed by measuring the plasma membrane permeability to 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI; 1 μg/ml, ThermoFisher Scientific/Molecular Probes, Waltham, MA, USA) or to propidium iodide (1 μg/ml, Sigma-Aldrich, St. Louis, MO)67. Fluorescence (10,000 cells/sample) was measured by flow cytometry using a FACS Aria I flow cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA). All measurements were carried out in duplicate.