Investigating the Immunomodulatory Effects of MLFB on LPS-Activated RAW 264.7 Cells

After RAW 264.7 cells were treated with LPS and different concentrations of MLFB for 24 h, the mRNA expressions of IL-1β, IL-6, TNF-α, NF-κB, PGE₂, TLR-4 in RAW 264.7 cells were determined by RT-qPCR (6 (link)). Firstly, TRIzol reagent was used to extract the total RNA of cells. Then RNA was synthesized into cDNA by cDNA reverse transcription kit referring to the manufacturer's descriptions. Finally, the RT-qPCR was proceeded in 20 μL reaction system containing 2 μL cDNA, 1 μL primer pairs (10 μmol/L), 10 μL SYBR Green PCR Master Mix, 0.4 μL 50 × ROX Reference Dye 2 and 6.6 μL ultra-pure distilled water. The PCR conditions were as followings: initial denaturation at 95°C for 5 min, 40 cycles at 95°C for 10 s, 60°C for 34 s, and the melting curve was obtained at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s in the ABI 7500 real-time fluorescent quantitative PCR System (Applied Biosystems, Foster, CA, USA). β-actin was used as internal control and the primers used here are shown in Table 1.