THP-1 cell lines treated with or without BTC and TLR2/4 ligands, and their subsequent gene expression

THP-1 cells treated with or without BTC and TLR2/4 ligands THP-1 monocytes were cultured in RPMI 1640 medium adjusted to contain 4.5 g/L glucose and supplemented with 1% Penicillin/Streptomycin, 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES. For experiments, monocytes were first seeded in 24 well plates at 400,000 cells/well in 1 mL of complete medium with 50 ng/mL PMA (phorbol 12-myristate 13-acetate) to induce polarization, for 24 hours. Then, attached cells were washed with sterile PBS to remove residual serum and PMA containing media. Cells were stimulated with a TLR2 agonist at 40 ng/mL (PGN-BS, Invivogen, San Diego, CA), TLR4 agonist at 4 ng/mL (LPS-B5, Invivogen, San Diego, CA), BTC at 40 ng/mL (human recombinant Betacellulin protein, R&D systems, MN), or combinations of all for 6 hours. Treatments were prepared in serum-free semi-complete RPMI 1640 media (see above for other components) supplemented with 20 μM BSA and 50 μM BHT (Butylated hydroxytoluene). Cells were lysed with 300 μL RLT buffer (Qiagen) and cell lysates were stored at -80 ºC.