4.7. DCF Assay

DCF assays were performed to measure the production of free radicals in the liver. This was carried out by referring to the method reported by Ali et al. [51], Puntel et al. [52], and Sugasawa et al. [53], with some modifications, adapted to the liver homogenate. First, the liver tissues (approximately 100 mg) were homogenized with a bead crusher in a protein lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5)) that included a protease inhibitor cocktail (Cat# 25955-24; Nacalai Tesque) in a micro-tube. Then, the homogenates were centrifuged at 12,000× g at 4 °C for 15 min. The supernatants of the protein lysate were transferred to new micro-tubes, and the protein concentrations were measured using a BCA assay kit (Cat#RR036A, Takara Bio, Kusatsu, Shiga, Japan) in accordance with the manufacturer’s instructions. After diluting and adjusting the protein lysate to 0.1 mg/mL protein, 10 µL of the protein lysate was mixed with 100 μL of 10 mM Tris-HCl (pH 7.4) containing 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in a black microplate well. After incubating this plate at 37 °C for 60 min, the fluorescence intensity of the solution at 525 nm was measured in duplicate by irradiating it with excitation light at 488 nm. The DCF concentrations were calculated from a regression equation plotted with a DCF standard (200 to 6.25 pmol/mL). Finally, the quantified values were normalized as DCF pmol/mg protein. This assay was conducted using duplicate measurements.