TLR7/8 agonist 1

The induction of NF-κB is quantified using human TLR-2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, and NOD-1/NOD-2-specific, rapid-throughput, liquid handler-assisted reporter gene assays. HEK293 cells stably co-transfected with the appropriate hTLR (or NOD) and secreted alkaline phosphatase (sAP) are maintained in HEK-Blue Selection medium. Stable expression of secreted alkaline phosphatase (sAP) under control of NF-κB/AP-1 promoters is inducible by appropriate TLR/NOD agonists, and extracellular sAP in the supernatant is proportional to NF-κB induction. Reporter cells are incubated at a density of ~10⁵ cells/mL in a volume of 80 μL/well, in 384-well, flat-bottomed, cell culture-treated microtiter plates in the presence of graded concentrations of stimuli. sAP is assayed spectrophotometrically using an alkaline phosphatase-specific chromogen (present in HEK-detection medium) at 620 nm^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Fresh human peripheral blood mononuclear cells (hPBMC) are isolated from human blood obtained by venipuncture with informed consent and as per institutional guidelines on Ficoll–Hypaque gradients. Aliquots of PBMCs (10⁵ cells in 100 μL/well) are stimulated for 12 h with graded concentrations of test compounds (e.g., TLR7/8 agonist 1; 0.1, 1, 10, and 100 μg/mL). Supernatants are isolated by centrifugation and are assayed in duplicates using analyte-specific multiplexed cytokine/chemokine bead array assays^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.