An Assay for TLR-Dependent NF-кB/AP-1 Transcription Factor Signaling in Macrophages

To assess the effects of an adsorbed protein layer on toll-like receptor-mediated NF-kB/AP-1 activity, after growing reporter macrophages in an appropriately-sized flask to 70% confluence, detach the cells with an appropriate enzymatic dissociation solution as demonstrated.

Resuspend the cells at a 7.3 x 10⁵ cells per milliliter concentration in assay medium and aliquot the cells evenly between three tubes. Treat the first tube of cells with one microgram per milliliter of TLR4 inhibitor for 60 minutes at room temperature. Treat the second tube with 50 micrograms per milliliter of anti-TLR2 antibody for 30 minutes at room temperature, and leave the third tube at room temperature without treatment.

While the cells are incubating, add 200 microliters of lysate and 10% fetal bovine serum to three sticky wells per condition. Allow the proteins to adsorb at 37 degrees Celsius for the appropriate experimental period before aspirating the protein solutions with a new Pasteur pipette for each well, and washing the sticky well surfaces three times with 250 microliters of PBS per well, for 5 minutes per wash.

At the end of the reporter macrophage treatment, add 200 microliters of cell solution to each well. Add Pam3CSK4 to a final concentration of 150 nanograms per milliliter to two wells as a TLR2-positive control as illustrated in the schematic.

For a TLR4-positive control, add lipopolysaccharide to a final concentration of 1.5 micrograms per milliliter to two wells. After 20 hours at 37 degrees Celsius, plate 20 microliters of supernatant from each well in duplicate in a 96-well plate, including three wells with 20 microliters of assay medium per well as the background control.

Next, add 200 microliters of SEAP reporter assay reagent to each well, and cover the plate with an adhesive seal for a 2 and 1/2 hour incubation at 37 degrees Celsius. Transfer the remainder of the supernatant to one 1.5-milliliter tube per sticky well, and sediment the debris by centrifugation. Then, transfer the supernatants to new 1.5-milliliter tubes for minus 80 degrees Celsius storage for downstream proinflammatory cytokine analysis by ELISA. At the end of the incubation, remove the adhesive seal from the plate and read the absorbance on a plate reader at 635 nanometers.