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.
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