*3.10. NO Production*

The nitrite levels released in the supernatant of the cultured RAW 264.7 cells were quantified to evaluate the immunostimulatory effect of the SPs of *C. cupressoides*, as described previously [66]. Initially, the cells were cultured (3 × 10<sup>5</sup>/well) in 24-well plates and exposed to different concentrations of the purified SPs (12.5–100 μg/mL) for 24 h. LPS (2 μg/mL) was used as a positive control. After the treatment time, 100 μL of the supernatant was collected and mixed with 100 μL of Griess reagen<sup>t</sup> and incubated for 10 min at room temperature in the dark. Absorbance was measured at 540 nm with a microplate spectrophotometer. Sodium nitrite was used as a standard, and the results were expressed as the percentage of nitrite production in relation to that of the positive control (LPS) according to the formula: % of nitrite production = (Atest/ALPS) × 100, in which Atest corresponds to absorbance of the experimental group and ALPS corresponds to the absorbance of LPS (positive control).

#### *3.11. Intracellular ROS Production*

The levels of intracellular oxygen reactive species were evaluated by quantifying the fluorescence emitted by 2,7-dichlorofluorescein, the oxidized form of 2,7-dichlorofluorescein diacetate (DCFH-DA). For this, RAW 264.7 macrophages were cultured (3 × 10<sup>5</sup>/well) in 24-well plates and exposed to SPs for 24 h. LPS (2 μg/mL) was used as a positive control. After treatment, the supernatant was discarded, cells were washed with phosphate buffered saline (PBS), and 100 μM DCFH-DA in DMEM containing 1% FBS was added, followed by incubation at 37 ◦C for 2 h. Then, the DCFH was removed, the cells were washed twice with PBS, and the emitted fluorescence was measured on a flow cytometer (FACSCanto II, BD Biosciences, Eugene, OR, USA) with FACSDiva software, version 6.1.2 (Becton Dickson, Franklin Lakes, NJ, USA). The results were analyzed in FlowJo software (FlowJo, Ashland, OR, USA) and expressed as % of fluorescence emitted relative to that of LPS-treated cells.
