Intracellular ROS Measurement in BV-2 Cells

Intracellular reactive oxygen species (ROS) levels were measured using the DCFDA cellular ROS detection kit (Abcam, Cambridge, UK). After internalization and subsequent hydrolysis, the redox indicator probe carboxy-H₂DCFDA is converted to carboxy-H₂DCF, which, in the presence of oxidant species, is converted to fluorescent carboxy-DCF [57 (link)]. BV-2 cells were seeded in black clear-bottom 96-well plates at a density of 5 × 10⁴ cells/well [43 (link)]. Cells were allowed to adhere overnight and then incubated with 20 μM DCFDA for 40 min at 37 °C in the dark. The solution was removed, and the cells were incubated in serum-free medium, containing LPA in the absence or presence of the antagonists for 3 and 6 h. Fluorescence intensity was measured with excitation and emission wavelengths of 485 and 535 nm, respectively.