2.8. RAW264.7 Macrophage Proliferation, and Nitric Oxide (NO) Production Assay

In the macrophage cell proliferation assay, RAW264.7 cells were determined according to the WST-1 method. The RAW264.7 cells (100 µL) were plated into a 96-well plate at 1 × 10⁶ cells/well in RPMI-1640 medium containing 10% FBS. The samples (MP, MFP1, and MPF2) were added at different concentrations (50, 100, and 200 µg/mL), and the medium was used as the control. The 96-well plate was incubated at 37 °C with 5% CO₂ for 24 h. After incubation, 10% WST-1 solution was added to each well, and the plate was further incubated for 1 h. The absorbance was determined using a microplate reader, and measured at 450 nm (EL-800; BioTek Instruments, Winooski, VT, USA).

The NO production assay was conducted by the Griess method [35]. In brief, RAW264.7 cells (1 × 10⁶ cells/well) were seeded in a 96-well plate, and treated with polysaccharides for 24 h. Treatment with lipopolysaccharide (LPS; 1 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) served as a positive control. After incubation, an equal volume of the Griess reagent was mixed with the supernatants. The absorbance was read at 540 nm using a microplate reader. NO production was calculated using a standard curve.