HEK-Blue hTLR4 Cells

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).