Hek blue null2 cells

RAW-Blue™ cells (In vivoGen) were grown in DMEM medium supplemented with 10% heat-inactivated FCS (30 min at 56°C), 100 U/ml penicillin/streptomycin, 2 mM glutamine, 100 μg/ml Normocin, and 200 μg/ml Zeocin for selection.
THP1-XBlue™ cells (In vivoGen) were grown in RPMI-1640 supplemented with 10% heat-inactivated FCS (30 min at 56°C), 100 U/ml penicillin/streptomycin, 2 mM glutamine, 100 μg/ml Normocin, and 200 μg/ml Zeocin for selection. For macrophage differentiation, 50,000 cells per well were cultured in 96 well plates in the presence of 30 ng/ml phorbol 12-myristate 13-acetate (PMA, Calbiochem #524400) for 48 hours.
HEK-Blue™ hTLR4 cells and HEK-Blue™ Null2 cells (In vivoGen) were grown in DMEM supplemented with 10% heat-inactivated FCS (30 min at 56°C), 100 U/ml penicillin/streptomycin, 2 mM glutamine, 100 μg/ml Normocin, and 1x HEK-Blue™ Selection.

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).