2.2.3. ROS Production in Neuro-2a Cells Subjected to Oxidative Stress after Exposition to Urolithin A

Neuro-2a cells were seeded in a 96-well plate. After 24 h, the medium was replaced with PBS supplemented with glucose and DCFH-DA (2,7-di-chloro-dihydrofluorescein diacetate, 0.01 M) for 30 min at 37 ◦C. After this time, PBS was removed and cells were washed twice with new PBS and treated with different concentrations of urolithin A (0.5–4 μM), as well as hydrogen peroxide (250 μM). The absorbance was checked at 480 nm (λexcitation) and 520 nm (λemission) wavelengths [14]. The kinetic was performed over 90 min in a Synergy H1 Hybrid Multi-Mode Reader (Winooski, VT, USA). Results were represented as a percentage of intracellular ROS production (100% of control).

2.2.4. Lipid Peroxidation in Neuro-2a Cells Subjected to Oxidative Stress after Exposition to Urolithin A (TBARS assay)

Lipid peroxidation was assessed using the protocol by Mitsuru Uchiyama [15]. First, 100 μL of thawed pellets were placed into 200 μL of TBA–TCA–HCl cocktail and subsequently boiled at 100 ◦C for 10 min to accelerate the reaction. The reaction was broken by placing the samples on ice and stunning them in a vortex three times for 20 min and centrifuged at 4 ◦C (3000 rpm, 10 min). The obtained supernatant was placed in three different wells of a 96-well plate and absorbance was read at 530 nm using a Synergy H1 Hybrid Multi-Mode Reader.

2.2.5. Activity of Antioxidant Enzymes in Neuro-2a Cells Subjected to Oxidative Stress after Exposition to Urolithin A

Before analyzing the activity of antioxidant enzymes, the protein content of Neuro-2a cells was calculated by colorimetric bicinchoninic acid (BCA) assay and normalized lysis buffer for 20 min (Ethylenediaminetetraacetic acid (EDTA) 1 mM, Tris 25 mM, NaCl 150 mM and 0.1% Triton; pH = 7.4). Moreover, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) proteinase inhibitors (20, 10, and 35 μL/mL, respectively) were aggregated into the buffer. Finally, supernatant was kept for the experiments and the precipitate was discard.

The activity of catalase was measured as follows: hydrogen peroxide (1970 μL, 15 mM) was prepared in sodium phosphate buffer (pH = 7.5) supplemented with 30 μL of supernatant [16]; the mixture was prepared in a quartz cuvette, measuring the absorbance for 30 secs at 240 nm, using a Shimadzu Spectrophotometer UV-1800 (Duisburg, Germany). The activity of the enzyme was expressed following the next equation: Catalase activity = ((ΔAbs/min) × 2 × F)/(0.0436 × Vs × C)

43.6 mL nmol−<sup>1</sup> cm<sup>−</sup>1: molar extinction coefficient of H2O2

F: dilution factor

C: Protein concentration (mg/mL)

Vs: Sample volume (mL)

ΔAbs/min: activity of the kinetic

For superoxide dismutase (SOD), a mixture of 1555 μL of Tris–DTPA buffer (50 mM, pH 8.2), 20 μL of pyrogallol (23.78 mM) diluted in HCl (10 mM), and 25 μL total cell extracts was placed in a quartz cuvette to measure the oxidation of pyrogallol at 420 nm for 1 min [17], using a Shimadzu Spectrophotometer UV-1800 (Duisburg, Germany). The activity of SOD was expressed with the following equation: % Inhibition = (ΔAbs control − ΔAbs sample)/(ΔAbs control) × 100

SOD activity = (% Inhibition × 2 × F)/(50 × Vs × C)

Glutathione reductase activity (GR) was quantified with the protocol developed by Staal et al. [18]: 1180 μL of 50 mM phosphate buffer\_\_EDTA (6.3 mM, pH 7.4), 50 μL of total cell extracts, 35 μL of glutathione disulfide (GSSG) 5 mM, and 35 μL of nicotinamide adenine dinucleotide phosphate (NADPH; 2.4 mM), which were mixed in a quartz cuvette. Glutathione peroxidase activity (GPx) was detected by mixing 1220 μL of 50 mM phosphate buffer\_\_EDTA 6.3 mM, 20 μL of total cell extracts, and 20 μL of glutathione (GSH) 10 mM. After 5 min in dark conditions, 20 μL of NADPH (2.4 mM) and 20 μL H2O2 63.5 mM were added to the cuvette [19,20]. GR activity was read for three minutes with 60 sec of delay at 340 nm using a Shimadzu Spectrophotometer UV-1800 (Duisburg, Germany). GPx activity was measured for 3 min at 340 nm and expressed in UI/mg protein. GR activity = ((ΔAbs/min) × 1.3 × F)/(0.00622 × Vs × C) GPx activity = ((ΔAbs/min) × 1.3 × F)/(0.00622 × Vs × C)

0.00622 mL nmol−<sup>1</sup> cm<sup>−</sup>1: molar extinction coefficient of NADPH

F: dilution factor

C: Protein concentration (mg/mL)

Vs: Sample volume (mL)

ΔAbs/min: activity of the kinetic
