TLR-Agonist Stimulation of Immune Cells

SCC24, SCC90, SCC72, SCC89 were stimulated with various concentrations of lipopolysaccharide (LPS: a non-specific TLR4 agonist) from Escherichia coli 0111:B4 (Sigma-Aldrich, St. Louis, MO, USA), lipopolysaccharide ultra-pure (LPS-UP: a specific TLR4 agonist) from E. coli (In vivo Gen, San Diego, California, USA), and peptidoglycan from Staphylococcus aureus (PGN: a TLR2 agonist) (Sigma-Aldrich, St. Louis, MO, USA). For ELISA, cells were stimulated with 0.01–1 µg/mL of LPS; 0.01-1 µg/mL of LPS-UP and 0.1–10 µg/mL of PGN [25 (link), 56 (link)]. For qPCR and Western blot, cells were stimulated with 1 µg/mL of LPS or LPS-UP or PGN. All incubations were undertaken in a humidified incubator (5% CO2) at 37^°C for 24 h. Experiments were undertaken using triplicate biological repeats.