This protocol enables the rapid assessment of NF-kappa-B and AP-1 activity in reporter macrophages cultured on adsorbed protein layers and the contribution of cell signaling pathways, like Toll-like receptors, to that response. The main advantage of this technique is its simplicity, allowing cell culture supernatants to be quickly analyzed for NF-kappa-B and AP-1 activity using a straightforward enzymatic assay. Begin by dissolving PMMA in chloroform at a 20 milligrams per milliliter final concentration in a 20-milliliter glass scintillation vial in a fume hood.
Place a magnetic stir bar in the vial, and stir the mixture for at least two hours. When all of the solids have been dissolved, transfer 400 microliters of the PMMA solution onto the center of each borosilicate glass microscope slide per planned condition on a spin coater, and spin the PMMA onto the slide for two minutes at 3, 000 rotations per minute. Then store the spin-coated slides in a 70%ethanol-sprayed clean box.
Next, in a biological safety cabinet, use sterile forceps and aseptic technique to attach eight-chamber sticky wells to the PMMA coated-slides, pressing firmly on the top of each sticky well to make sure they are strongly attached. Take care to line up the sticky wells with the edges of the microscope slide so that all of the wells attach to the slide and don't leak. Incubate the sticky well-attached slides at 37 degrees Celsius overnight to secure the seals.
Then add 200 microliters of cell culture grade endotoxin-free water to each well for a 60-minute incubation at room temperature to test the seals the next morning. At the end of the incubation, aspirate the water, taking care not to disturb the PMMA coating. Wash each well three times with 300 microliters of fresh endotoxin-free water for one hour per wash followed by one 12-hour and one 24-hour wash prior to use to remove any remaining solvent.
Then UV sterilize the slides for 30 minutes. To obtain 3T3 cell lysates, when the 3T3 cell cultures reach 70%confluence in T150 culture flasks, wash each culture with five milliliters of PBS, and treat the cells with five milliliters of animal origin-free, recombinant cell dissociation enzyme per flask at 37 degrees Celsius for three to five minutes. At the end of the incubation, gently tilt each flask back and forth a few times to detach the cells before neutralizing the enzyme with five milliliters of PBS per culture.
Pool the dissociated cells in a single 50-milliliter centrifuge tube, and use a pipette to break up any cell clumps. After counting, collect the cells by centrifugation. Aspirate the supernatant, and resuspend the cells at a one times 10 to the six cells per milliliter concentration in fresh PBS.
Then freeze the cells in a minus 80-degree Celsius freezer for at least two hours until the sample is fully frozen before placing the frozen cell solution in a 37-degree Celsius water bath until completely thawed. To assess the effects of an adsorbed protein layer on Toll-like receptor-mediated NF-kappa-B/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 times 10 to the five 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 five 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 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-degree 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 plater reader at 635 nanometers. Soaking PMMA-coated microscope slides in 70%ethanol for one hour removes the PMMA coating, while neither 70%ethanol nor UV sterilization influences the water contact angle of fPTFE-coated coverslips.
Western blot analysis of 3T3 lysates reveals the presence of both HMGB1 and heat shock protein 60, two well-documented damage-associated molecular patterns. The adsorption of TLR ligands from the lysate onto the polymer surfaces can be confirmed by culturing reporter macrophages for 20 hours on the protein-adsorbed polymer surfaces and then indirectly assessing the NF-kappa-B/AP-1 activity via an enzymatic assay. This activity is reduced following inhibition of TLR2 or TLR4 signaling.
Further, reporter macrophages have significantly increased NF-kappa-B/AP-1 activity in response to adsorbed lysate compared to adsorbed FBS or plasma and no pre-adsorbed protein. In addition, small amounts of lysate diluted in serum induce significantly increased NF-kappa-B/AP-1 responses compared to serum alone, with the lowest effective dilution dependent on the polymer surface. To avoid endotoxin contamination of the coated surfaces, work in clean areas, cover the surfaces when not in use, and use cell culture grade water and buffers for rinses.
Following this procedure, immunoassays can be performed on the remaining supernatants to assess protein secretion, and macrophages can be analyzed by flow cytometry and qPCR gene expression analysis.
