Quantifying Intracellular and Tissue ROS Levels

Intracellular ROS levels were measured in B35 cells (neural neuroblast cells that originate from the neuroblastoma of BDIX rats) by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich Co.), as previously described [22 (link)]. Briefly, B35 cells were seeded at 5 × 10⁵ cells/2 mL in six-well plates and incubated with 100 μg/mL of GEGR for 1 h at 37 °C. After washing with 1× PBS, the cells were incubated with H₂O₂ (100 μM; Junsei Chemical Co.) for 24 h. Next, the stimulated cells along with 25 μM DCFH-DA were incubated for 30 min at 37 °C. Finally, the cells were washed twice with 1× PBS, and the presence of green fluorescence was observed under a fluorescence microscope (200× magnification; Eclipse TX100, Nikon, Tokyo, Japan).
ROS level in the brain cortex tissue was assessed by employing a proprietary quenched fluorogenic probe and the reagents provided in the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs Inc., San Diego, CA, USA). Briefly, the brain cortex tissue samples (100 mg) were homogenized in 600 μL of 1× PBS using a glass homogenizer. Each sample and standards (50 μL) were mixed with Catalyst (50 μL) in 96-well plate for 5 min. DCFH solution (100 μL) were added into this mixture and incubated under the light protection for 15–45 min at room temperature. The final fluorescence of each well were measured at 480 nm excitation/530 nm emission.