2.9. mRNA Expression of Cytokines by RT-PCR and Quantitative Secretions by ELISA Kits

The total RNA was extracted from HD11 cells by applying Trizol (TransGen Biotech, Beijing, China), and was subsequently treated with a DNA-free kit to filter DNA contamination. Relative quantification of interleukin (IL)-1β, IL-2, IL-6, IL-10, Interferon gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) was performed using an SYBR Green PCR Master Mix kit (Takara, Dalian, China), as previously described [15]. As for cytokine determination, roughly 200 μL aliquots of each sample were used to measure the cytokines IL-1β, IL-2, IL-6, IL-10, IFN-γ, and TNF-α with commercial ELISA kits (Kingfisher Biotech Inc., Saint Paul, MN, United States).