Quantifying Inflammatory Cytokine Responses

BMMs (1 × 10⁶ cells/well, n = 4 per group) were seeded into six-well culture plates (Falcon) to perform the qRT-PCR analyses. Cells were treated with each concentration of MP (1 μg/mL) and TEGO (100 μg/mL) containing LPS (0.1 μg/mL). After 24 h, total RNA from seeded cells were extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The qRT-PCR analyses were performed as described previously [8 (link),33 (link),34 (link)]. The primers were obtained from Bioneer (Daejeon, Republic of Korea). The relative gene expression values of IL-6, IL-1β, TNF-α, IL-4, IL-10, and TGF-β were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by using the 2-ΔΔCT calculation [37 (link)]. A detailed description of the qRT-PCR method, including the nucleotide sequences of primers, is provided in the Supporting Information.