Enzyme substrate

HEK cell line expressing TLR4 and its cofactors MD2 and CD14 were also stably transfected with SEAP (secreted embryonic alkaline phosphatase), as reporter gene, under the control of NF-κB promoter. These cells were purchased from InvivoGen. In this model, the activation of NF-κB can be monitored by a colorimetric assay quantifying the activity of the secreted SEAP in the cell supernatants in the presence of enzyme substrate as described by the manufacturer (InvivoGen).

HEK-Blue hTLR4 cells and HEK-Blue Null2 cells (as control) were purchased from InvivoGen (San Diego, CA, USA). The HEK-Blue hTLR4 cells were obtained by co-transfection of the human TLR4 (hTLR4) gene, the myeloid differentiation factor 2 (MD-2) and CD14 co-receptor genes, and a secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. The SEAP reporter gene is placed under the control of an IL-2 p40 minimal promoter fused to five NF-κB and AP-1 binding sites. Cells were grown in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 100 µg/mL Normocin with selection antibiotic and passaged when 70% confluence was reached.
The activation of NF-κB can be monitored by a colorimetric assay quantifying the activity of the secreted SEAP in the cell supernatants in the presence of enzyme substrate as described by the manufacturer (InvivoGen).