Stimulating NF-κB Activity in Macrophages

THP-1 cells and reporter cells were cultured according to a previously described protocol (Chen et al., 2020 (link)). Bone marrow-derived macrophages (BMDMs) were isolated from mice and differentiated for 5–6 days, following standard procedures (Hsu et al., 2015 (link)). THP-1 cells or BMDMs were treated with MDP fractions at the indicated concentrations for 6 h. The cells were then stimulated with 0.2 μg/ml Pam3CSK4 (tlrl-pms, InvivoGen) or 0.1 ng/ml LPS (SI-L8274, Sigma-Aldrich). After stimulation for 14–16 h, NF-κB activity was quantified by measuring the levels of alkaline phosphatase in the culture supernatant. Changes from baseline were calculated as -fold changes and normalized against unstimulated control cells.