N acetyl l cysteine nac c 2 mm

Disruption of MMP after PSE 45 µg/m and Phy 60 µM treatment (3, 24, 48 or 72 h) was analyzed by flow cytometry using 0.1 μM TMRE (Molecular Probes, Eugene, OR, USA) staining. NAC/PSE or NAC/Phy experimental groups were pre-treated with N-acetyl-L-cysteine (NAC c = 2 mM; Merck) for 1 h before PSE/Phy were applied. The stained cells were incubated for 30 min at room temperature in the dark and then washed twice with PBS, resuspended and analyzed (1 × 10⁴ cells per sample). Fluorescence was detected with 585/42 (FL-2) optical filter by flow cytometer (BD FACSCalibur). All experiments were performed in triplicate.

Flow cytometry was used to analyze intracellularly produced oxygen radicals detected by MitoSOX^TMRed mitochondrial superoxide indicator (Thermo Fisher) or dihydrorhodamine-123 (DHR-123, Merck), which reacts with intracellular hydrogen peroxide (ROS). Jurkat cells treated with PSE 45 µg/mL and Phy 60 µM were harvested 6, 24, 48 and 72 h after exposure, washed and resuspended in PBS. NAC/PSE or NAC/Phy experimental groups were pre-treated with N-acetyl-L-cysteine (NAC c = 2 mM; Merck) for 1 h before PSE/Phy were applied. The samples were incubated for 15 min in the dark with DHR-123 (0.2 µM) and MitoSOX red (5 µM) and, after incubation, were immediately placed on ice. Fluorescence was detected with 530/30 (FL-1) and 585/42 (FL-2) optical filters, respectively, by flow cytometer (BD FACSCalibur). A minimum of 1 × 10⁴ events were analyzed per analysis, and all experiments were performed in triplicate.