2.6. Protective Effects in Macrophage RAW264.7

The mouse macrophage cell line RAW264.7 (American Type Culture Collection, ATCC, Rockville, MD, USA) was grown in DMEM medium supplemented with 10% of FBS (v/v) and 1% penicillin/streptomycin (v/v). Cells were seeded in 75 cm² culture flasks and incubated at 37 °C in a humidified incubator containing 5% CO₂ and 95% air. Culture medium was changed every 2 days and subcultures were made by scraping.

Cell viability was determined using the MTT assay. RAW264.7 cells were seeded onto 96-well plates at a density of 6 × 10⁴ cells/well in complete medium with 10% FBS and incubated for 24 h at 37 °C. After removing the culture medium, samples were added. In the case of lipopolysaccharide (LPS)-stimulated cells, 20 μL of LPS (100 ng/mL, final concentration) was also added. Once incubated for 24 h at 37 °C, the supernatant was removed, and cells were washed with PBS. Then, an MTT solution (5 mg/mL in PBS) was added and the plate was incubated for 120 min at 37 °C. Once the supernatant was aspirated, insoluble formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm in the Multiskan FC plate reader (ThermoTM Scientific, Wilmington, DE, USA). The results were expressed as percentage of the control, considered as 100%.

Intracellular ROS levels were quantified according to the method described by LeBel et al. [23] using dichlorofluorescein (DCFH-DA) as probe. RAW264.7 macrophages were seeded onto 48-well plates (4.75 × 10⁴ cells/well) in complete medium with 10% FBS and incubated for 24 h at 37 °C. Once the medium was aspirated, samples were added and cells were incubated for 24 h at 37 °C. In stimulated cells, 20 μL of LPS (100 ng/mL) was also added. After aspirating the supernatant, 100 µL of a solution containing 5 mM DCFH-DA dissolved in Hank’s balanced salt solution (HBSS, Sigma-Aldrich) was added to the wells, incubating the plate at 37 °C for 60 min. The fluorescence was measured in a FLUOstar OPTIMA plate reader (BMG Labtech) at λ_excitation of 485 and λ_emission of 530 nm. The results were expressed as ROS levels (% compared with the control, considered as 100%).

RAW264.7 macrophages were seeded onto 96-well plates (1× 10⁵ cells/well) in complete medium with 10% FBS and incubated for 24 h at 37 °C. Once the medium was aspirated, samples were added (100 μL) and cells were incubated for 24 h at 37 °C. A volume of 20 μL of LPS (100 ng/mL) was added to stimulated cells. Nitrite accumulation, and indicator of nitric oxide (NO) synthesis, was measured in the macrophage culture medium by the Griess reaction following a previously described method [24]. Briefly, a mixture containing 100 μL of supernatant and 100 μL of Griess reagent [1% (w/v) sulfanyl amide and 0.1% (w/v) N-1-(naphthyl) ethylenediamine-di-HCl in 2.5% (v/v) H₃PO₄] was incubated for 15 min and the absorbance measured at 550 nm in a Synergy HTX microplate reader (BioTek Instruments, Inc.). A sodium nitrite standard curve (3.125–100 μM) was used to measure the amount of NO. Three independent experiments were conducted and data were expressed as the mean and standard deviation (SD) (n = 12).