Oxidative stress biomarkers were analyzed in lymphocytes and monocytes using the single-laser FACSCalibur (BD Biosciences) cytometer. Test standardization and data acquisition analysis were performed using the CELL Quest software (BD Biosciences). A forward and side scatter gate was used for the selection and analysis of the different cell subpopulations.
For the assessment of reactive oxygen species (ROS) generation, including peroxides and peroxynitrites, cells were incubated with 20.5 µM dichloro-dihydro-fluorescein diacetate (DCFHDA; Sigma-Aldrich, St Louis, MI, USA) and 5 µM dihydrorhodamine-123 (DHRH-123; Sigma-Aldrich) for 30 min in the dark at 37 °C. For the detection of intracellular glutathione (GSH), cells were incubated with 1 µM 5-chloromethylfluorescein diacetate (CMF-DA; Invitrogen, Eugene, OR, USA) for 30 min in the dark at 37 °C. Then, cells were washed, resuspended in PBS, and analyzed on a single-laser FACSCalibur (BD Biosciences) cytometer. The JC1 Mitoscreen assay (BD Biosciences) was used (final concentration 2 µM) to assess mitochondrial membrane potential (ΔΨm) according to the manufacturer’s instructions.
Mitochondrial superoxide dismutase (SOD2), catalase, and glutathione peroxidase (GPx) activities were assayed in cell lysates using specific kits (Cayman Chemical) following the manufacturer’s recommendations.
For the assessment of reactive oxygen species (ROS) generation, including peroxides and peroxynitrites, cells were incubated with 20.5 µM dichloro-dihydro-fluorescein diacetate (DCFHDA; Sigma-Aldrich, St Louis, MI, USA) and 5 µM dihydrorhodamine-123 (DHRH-123; Sigma-Aldrich) for 30 min in the dark at 37 °C. For the detection of intracellular glutathione (GSH), cells were incubated with 1 µM 5-chloromethylfluorescein diacetate (CMF-DA; Invitrogen, Eugene, OR, USA) for 30 min in the dark at 37 °C. Then, cells were washed, resuspended in PBS, and analyzed on a single-laser FACSCalibur (BD Biosciences) cytometer. The JC1 Mitoscreen assay (BD Biosciences) was used (final concentration 2 µM) to assess mitochondrial membrane potential (ΔΨm) according to the manufacturer’s instructions.
Mitochondrial superoxide dismutase (SOD2), catalase, and glutathione peroxidase (GPx) activities were assayed in cell lysates using specific kits (Cayman Chemical) following the manufacturer’s recommendations.