2.4.3. Nitric Oxide (NO) and Interleukin 6 (IL-6) Productions from RAW264.7 Cells

The formation of nitrites in the culture medium of macrophages, stimulated with MP-PSC, was quantified using the Griess reagent (Abcam, Cambridge, UK) [57]. The nitrites served as an indirect indicator for NO production. RAW264.7 cells (TIB-71, American Type Culture Collection, Manassas, VA, USA) were cultured in 12-well flat-bottom plates at a cell density of 2.5 × 10⁵ cells/well with 100 µL of MP-PSC (50, 100, and 200 μg/mL), dissolved in DMEM-high Glc growth medium, containing 10% (v/v) FBS, and 1% (v/v) penicillin/streptomycin (HyClone^TM, Cytiva, Marlborough, MA, USA). The incubation was performed for 24 h at 37 °C and with a CO₂ supply of 5%. The final reaction volume was 300 μL. Additionally, the macrophages were co-treated with MP-PSC and 10 ng/mL LPS (E. coli/026:B6) under the same conditions. After 24 h, the supernatants were collected by centrifugation at 16,000× g at 4 °C for 5 min. Eighty microliters of each sample was mixed with 80 μL of the Griess reagent, and the reaction mixtures were incubated in 96-well plates at RT for 30 min in the dark. Absorbance measurement was performed at 540 nm (SPECTRA Sunrise^TM microplate reader, Tecan, Männedorf, Switzerland). For the preparation of a calibration standard curve, 0–52 μM NaNO₂ solutions, prepared in DMEM, were used. Data were converted to a percentage of the control or LPS control, and they were presented as the mean ± SEM (n = 3) from three separate runs.

Simultaneously, IL-6 production by RAW264.7 cells, treated with MP-PSC, LPS and MP-PSC+LPS, was also determined after 24 h of exposure. The analysis was performed according to an immunoassay kit Mouse IL-6 DuoSet (R&D Systems, Minneapolis, MN, USA). Data from three independent runs were converted to a percentage of the control (mean ± SEM, n = 3).