Evaluating THP-1 and HEK-Blue Responses to Immune Stimuli

THP-1 cells were seeded in 96-well plates (Corning Costar) at a density of 100,000 cells per well. First, cells were stimulated for 18 h with rSIP, FLH, LPS, a NOD 1 ligand (positive control for NF-kB; C12-iE-DAP, InvivoGen), and a PRR agonist (positive control for IRF; Poly (dA:dT)/LyoVec™, InvivoGen). The supernatant was then subjected to a colorimetric enzyme assay to measure alkaline phosphatase (AP) activity using the commercial QUANTI-Blue™ solution (InvivoGen). The supernatant was then incubated at 37°C for 3 h, and the optical density was read at 650 nm in an Epoch 2 reader (BioTek). On the other hand, luciferase activity (LUCIA) was measured using the commercial solution QUANTI-Luc ™ (InvivoGen), which has a coelenterazine substrate and stabilizing agents for the luciferase reaction. The light signal produced was then quantified using a Berthold luminometer (Model LB9515), and the signal was expressed as relative light units (RLUs).
Hek-Blue cells (hkb-mtlr4 and hkb-htlr4, InvivoGen) express SEAP under the control of promoters containing binding elements for the NF-κB transcription factor (35 (link)). Hek-Blue cells were seeded in 96-well plates (Corning Costar) at a density of 25,000 cells per well in HEK-Blue™ Detection medium (InvivoGen). Then, the cells were stimulated for 48 h with rSIP, FLH, and LPS, and SEAP was quantified using an Epoch 2 reader (Biotek).