Macrophage activation assay

RAW 264.7 cells were purchased from ATCC (CAT# TIB-71) and NFκB reporter THP-1_Lucia human cell lines were purchased from InvivoGen (CAT#: thpl-nfkb) employed for downstream experiments of this study. Passages 5–10 were cultured in RPMI 1640 supplemented with 10% of heat-inactivated FBS in the presence of 1% penicillin/streptomycin and 1% L-glutamine. Cells were then stimulated with 10 or 100 ng/mL of LPS (Invitrogen, CAT#: 50-112-2025). Stimulated and non-stimulated cells were then mixed with XOs with the mentioned concentrations in the results section. Control cells, LPS-stimulated cells in the presence and absence of XOs, and non-stimulated cells in the presence and absence of XOs (100,000 cells for each condition) were co-cultured for 10–14 h at 37 °C in a humidified incubator with 5% CO₂. Next, supernatant was collected for cytokine analyses. Supernatants were centrifuged at 2500 × g and 4 °C for 5 min and stored at −80 °C. Samples were then shipped on dry ice to Eve Technologies (Calgary, Canada), where cytokines were analyzed using Mouse Focused 32-Plex Discovery Assay (CAT#: 17619).