An Assay for TLR-Dependent NF-кB/AP-1 Transcription Factor Signaling in Macrophages
Transkript
Take tubes containing reporter macrophages with the gene for secreted embryonic alkaline phosphatase or SEAP enzyme under an inducible promoter.
In one tube, introduce an antibody specific for macrophage Toll-like receptor 2 or TLR2, neutralizing its activity.
In another, add an inhibitor to suppress TLR4 signaling. Leave the last tube untreated.
Take a chambered slide with an adsorbed layer of damage-associated molecular patterns or DAMPs — proteins released during cell necrosis.
Add treated and untreated macrophages in separate wells and incubate.
TLR2 and TLR4 on untreated macrophages bind to DAMPs, activating the transcription factors nuclear factor-kappa-B, or NF-κB, and activator protein-1, or AP-1.
These translocate to the nucleus and bind to the SEAP promoter, upregulating SEAP production, which is secreted extracellularly.
Transfer the SEAP-containing supernatant to a microplate with a chromogenic substrate.
SEAP hydrolyzes the substrate, producing a purple-blue-colored product. Measure the absorbance.
TLR2 neutralization strongly reduces SEAP production, indicating TLR2 as the primary mediator.
