Tlrl mdpc

For experiments, THP-1 cells were seeded at a density of 1 × 10⁶ cells/ml and differentiated into macrophages using 100 μg/ml PMA (Sigma-Aldrich, P1585) for 48 h. Medium was changed to serum-free medium 6 h before transfection with MDP (100 ng/ml; InvivoGen, tlrl-mdpc), or to serum-free medium containing ultrapure lipopolysaccharide (upLPS, 100 ng/ml; InvivoGen, tlrl-3pelps) 16 h before treatment with MSU (150 μg/ml; InvivoGen, tlrl-msu) or ATP (2 mM; InvivoGen tlrl-atp). For transfection, MDP (InvivoGen, tlrl-mdpc) was dissolved in DMSO and mixed with Lipofectamine® (InvivoGen, L3000–001) for 15 min before applying to the cell culture media. As a control, DSMO mixed with Lipofectamine® was used. MSU and ATP were applied onto the cells as suspensions. Likewise, BMDCs were collected at d 7 of BM cultures in differentiation medium, and seeded in new plates (0.5 × 10⁶ cells/well in 12-well plates) in RPMI supplemented with 10% FCS. After 6 h, medium was changed for serum-free medium 6 h before transfection with MDP or treatment with MSU or ATP as described above.