Tl 506

BM cells from B6.huTLR8tg mice or C57BL/6 littermates were plated in triplicates in 96-well plates stimulated with TLR7-specific agonist CL264 or TLR8-specific agonist TL-506 (InvivoGen) at the indicated concentrations for 24 h and then TNFα from the supernatants was measured by ELISA (R and D). 2 × 105 BMDM from TLR7-deficient mice were plated overnight in XF media on Seahorse culture plates that were pre-coated with Cell-Tak (Corning cell and tissue adhesive) for 20 min at RT, washed with H₂O and air dried before cell plating. Seahorse culture plates were pre-coated with Cell-Tak (25 μl of 11.2 µg/mL) for 20 min at RT (Corning cell and tissue adhesive), washed with H₂O, and air dried before cell plating. Cells were stimulated with agonists CL075 (3M002; InvivoGen) or TL-506 (InvivoGen) for 24 h. BMDM extracellular acidification rate was then measured using a Seahorse XF96 Extracellular Flux Analyzer and the Glycolysis Stress Test kit according to manufacturer’s instructions (Agilent). Data were normalized to the cell number.