Intracellular ROS Measurement in LPS-induced RAW 264.7 Cells

RAW 264.7 cells (1 × 10⁴), seeded on black/clear bottom 96-well-microtiter plate, were pre-treated with AJHLE at non-toxic concentrations (12.5, 25, and 50 µg/mL) for 2 h and then exposed or not to LPS (200 ng/mL) for 24 h under standard culture conditions. At the end of the experiment, the fluorescent probe 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, Hamburg, Germany), was used to evaluate the intracellular ROS levels [29 (link)]. Precisely, after washing the cells with PBS, fresh culture medium without FBS, and containing 10 µM of DCFH-DA, was added and further incubated under standard culture conditions for 30 min. After this period, the fluorescent probe was replaced with PBS, and the cell fluorescence was measured at 485 nm (excitation) and 535 nm (emission) with a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, USA). Intracellular ROS levels were expressed as percentages of negative control (cells treated with DMSO 0.5% without LPS). Images were captured using a Flexacam C1 camera (Leica Microsystems GmbH, Wetzlar, Germany) connected to a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) using an excitation/emission filter (480/535 nm) on 40X objective.