Macrophage Cytokine Expression Assay

RAW 264.7 macrophages were plated in 12-well plate (5 × 10⁵/well). Cells pretreated with the test samples, positive control (parthenolide, 10 μM), or solvent vehicle (0.1% DMSO in culture medium) for 1 h were incubated with 100 ng/mL LPS for additional 4 h. Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of RNA was performed with the Reverse Transcription System A3500 kit (Promega, Madison, WI) according to the manufacturer's protocol. Relative quantification of gene expression was performed with SYBR® Green Real-Time PCR Master Mix (TOYOBO, Osaka, Japan) and conducted with the Eppendorf Mastercycler ep realplex (Hauppauge, NY). The following primers were used: TNF-α (5′-TTCTCATTCCTGCTTGTGG-3′; 5′-ACTTGGTGGTTTGCTACG-3′), IL-6 (5′-CTTCTTGGGACTGAT G-3′; 5′-CTGGCTTTGTCTTTCT-3′), and IL-1β (5′-GATCCACACTCTCCAGCTGCA-3′; 5′-CAACCAACAAGTGATATTCTCCATG-3′). The primers for the mouse housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5′-CCTTCCGTGTTCCTACC-3′ and 5′-CAACCTGGTCCTCAGTGT A-3′ [9 (link)].