Vesicle Anti-Inflammatory Activity in Macrophages

The anti-inflammatory activity of the vesicles was assessed by inducing nitric oxide (NO) production in murine macrophages using LPS as previously described (Schulte-Werning et al., 2021 (link)). RAW 264.7 cells (Basnet et al., 2012 (link)) in complete RPMI medium [containing 10% (v/v) FBS and penicillin–streptomycin] were plated on 24-well plate (1,000 μl, 5 × 10⁵ cells/ml) and incubated (37°C, 5% CO₂) for 24 h. The complete medium was aspirated and LPS (1 μg/ml, 990 μl) in complete RPMI added to each well. Next, diluted vesicle suspensions (10 μl) were added to the wells at final lipid concentration of 1, 10, and 50 μg/ml, and the plates incubated for another 24 h (37°C, 5% CO₂). The NO production was assessed by mixing the cell medium and Griess reagent [1:1, v/v; 2.5% phosphoric acid with 1% sulphanilamide and 0.1% N-(−1-naphthyl)ethylenediamine] and analyzing the mixture with Spark M10 multimode plate reader (Tecan Trading AG, Männedorf, Switzerland) at 560 nm. Only complete medium or LPS (1 μg/ml) in complete RPMI served as controls. The LPS-induced cells treated with vesicles were compared to non-treated LPS-induced cells (100%).