The authors gratefully acknowledge operational funding from Canadian Institutes of Health Research Project (PTJ 162251), Queen&#39;s University Senate Advisory Research Committee and infrastructure support from the Canadian Foundation for Innovation John Evan&#39;s Leadership Fund (Project 34137) and the Ministry of Research and Innovation Ontario Research Fund (Project 34137). L.A.M. was supported by a Queen&#39;s University R. Samuel McLaughlin Fellowship, a Natural Sciences and Engineering Research Council of Canada Canadian Graduate Scholarship Master&#39;s Award and an Ontario Graduate Scholarship. The authors would like to thank Dr. Myron Szewczuk for his generous gift of the NF-&#954;B/AP-1 reporter macrophage cell line and Drs. Michael Blennerhassett and Sandra Lourenssen for the use of their gel imaging system and plate reader.

1. Media and Reagent Preparation
<ol>
	<li>Prepare fibroblast media. Combine 450 mL of Dulbecco&#39;s modified Eagle medium (DMEM), 50 mL of fetal bovine serum (FBS), and 5 mL of penicillin/streptomycin. Store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare reporter macrophage growth media in 50 mL aliquots. Combine 45 mL of DMEM, 5 mL of FBS, 5 &#956;g/mL mycoplasma elimination reagent (Table of Materials), and 200 &#956;g/mL phleomycin D1 (Table of Materials). Store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare reporter macrophage assay media in 50 mL aliquots. Combine 45 mL of DMEM, 5 mL of heat inactivated FBS (HI-FBS), 5 &#956;g/mL mycoplasma elimination reagent, and 200 &#956;g/mL phleomycin D1. Store at 4 &#176;C for up to 3 months.</li>
</ol>
2. Coating Cell Culture Surfaces with Poly(methyl methacrylate)
<ol>
	<li>Dissolve poly(methyl methacrylate) (PMMA) in chloroform at 20 mg/mL (e.g., 100 mg of PMMA in 5 mL of chloroform) in a 20 mL glass scintillation vial. Place a magnetic stir bar in the vial and allow to stir for at least 2 h, until all solids are dissolved. 
	CAUTION: Chloroform is harmful if inhaled. Ensure to use solvent in a fume hood while wearing PVA gloves.</li>
	<li>Pipette 400 &#181;L of PMMA solution onto the center of a borosilicate glass microscope slide in a spin coater, and spin at 3000 rpm for 2 min. Prepare the number of slides required for the assay, as well as 3&#8722;5 extra for water contact angle measurement. Store slides in a clean box (sprayed and wiped with 70% ethanol) for future use. 
	NOTE: Spin coating is often used to deposit a thin, uniform coating on a flat surface. A spin coater rotates a substrate at high speeds, using centrifugal force to spread the coating solution over the surface.
	<ol>
		<li>Measure water contact angle at two random positions on the surface of extra coated slides (i.e., not the slides being used for cell culture) with a goniometer to ensure glass surface was completely coated with the polymer. 
		NOTE: Only water of the highest purity (e.g., glass triple distilled) should be used for water contact angle measurements.</li>
	</ol>
	</li>
	<li>In a biological safety cabinet (BSC) attach 8-chamber sticky wells to PMMA coated-slides using sterile forceps and following aseptic technique. Press firmly on the top of the sticky wells to make sure they are strongly attached. Incubate the slides with attached sticky-wells at 37 &#176;C overnight to secure the seal.
	<ol>
		<li>Test the seal of the sticky wells by adding 200 &#181;L of cell culture grade (endotoxin-free) water to each well. Incubate at room temperature (RT) for 60 min and ensure no leakage before proceeding. Aspirate the water, being careful not to disturb the PMMA coating.</li>
	</ol>
	</li>
	<li>Perform endotoxin-free water washes by adding 300 &#181;L of endotoxin-free water to each well and incubating for 1 h (three times), 12 h, and 24 h prior to use to remove any remaining solvent.</li>
	<li>Test endotoxin concentration of the slides to be used for cell culture. Incubate 200 &#181;L of endotoxin-free reagent water (Table of Materials) in one well of each slide for 1 h. Measure endotoxin concentration in the extract using an endpoint chromogenic endotoxin assay (Table of Materials). 
	NOTE: The following protocol is specific to the endotoxin assay kit listed in the Table of Materials.</li>
	<li>Use only water and consumables (i.e., pipette tips, microcentrifuge tubes and well plates) that are certified pyrogen-free (i.e., endotoxin-free) for this work. Also, any glassware used in the preparation of the polymer-coated surfaces should be depyrogenated using dry heat sterilization (250 &#176;C for 30 min) prior to use10. Measuring endotoxin in the extract solution, as described here, can result in an underestimation of endotoxin on the material surface11,12. Consequently, it is recommended that when developing a polymer coating protocol, perform the endotoxin assay reaction (i.e., steps 2.5.4&#8722;2.5.6 for test samples [reagent water] or spike controls) directly within wells containing the coated sample to ensure no sources of endotoxin are inadvertently introduced into the system during the coating process.
	<ol>
		<li>Bring all test samples (i.e., extracts) and endotoxin assay reagents to RT. Reconstitute chromogenic reagent in assay buffer and endotoxin standard in reagent water, allow to dissolve for 5 min and gently swirl before using. Cover all bottles with paraffin film when not in use.</li>
		<li>Create a 5&#8722;8 point standard dilution curve of endotoxin standard ranging from the lower to the upper limit of the assay by performing a serial dilution of the endotoxin standard in reagent water.</li>
		<li>To control for enhancement or inhibition of the endotoxin assay in test samples, prepare a positive control (also called a spike control or spiked sample) by diluting a known amount of endotoxin in unused test sample solution. 
		NOTE: The concentration of the positive control should be the same concentration as a standard in the middle of the standard curve. If the recovered amount of the endotoxin spike (i.e., concentration of the positive control minus the concentration of the unspiked test sample) is within 50&#8722;200% of the nominal concentration of the endotoxin spike, the extraction solution can be considered to not significantly interfere with the assay.</li>
		<li>Add 50 &#181;L of standards, samples, or spike controls to each well of a 96-well plate in duplicate or triplicate. Use reagent water as a negative control.</li>
		<li>Add 50 &#181;L of chromogenic reagent to every well. Add reagent quickly to all wells. Use a timer to record the amount of time it takes to add reagent to all wells. Cover the plate with an adhesive seal and incubate at 37 &#176;C (incubation time is lot-dependent and stated on Certificate of Analysis included in the chromogenic reagent kit). Alternatively, check on the plate every 15 min during incubation until color change is observed in all standard wells.</li>
		<li>After incubation, add 25 &#181;L of 50% acetic acid to each well (final concentration of 10% acetic acid per well) to stop the reaction. Add acetic acid in the same order as the chromogenic reagent was added. Read absorbance of the plate using a plate reader at 405 nm. Aspirate liquid and discard plate. 
		NOTE: Acetic acid addition should take the same length of time to add to each well as the chromogenic reagent took (&#177; 30 s).</li>
	</ol>
	</li>
	<li>Ultraviolet (UV) sterilize the slides for 30 min prior to cell culture experiments.</li>
</ol>
3. Coating Cell Culture Surfaces with Polydimethylsiloxane
<ol>
	<li>Mix polydimethylsiloxane (PDMS) elastomer in a 10:1 weight ratio (base:curing agent). In a biological safety cabinet, pipette approximately 10 mL of polydimethylsiloxane base into a sterile tube. Weigh the tube and slowly add curing agent until 10% has been added. 
	CAUTION: Use PDMS reagents in a well-ventilated area and avoid eye contact by wearing safety glasses.</li>
	<li>Thoroughly mix the elastomer by stirring with a sterile serological pipette tip and by pipetting up and down. Add approximately 200 &#956;L of the solution to each well of a 48-well plate. Tilt the well plate slowly to ensure complete coverage of wells with elastomer solution.</li>
	<li>Place the well plate with elastomer into a vacuum oven set at 50 cmHg, 40 &#176;C. Remove the lid and cover with a single-ply wipe to prevent other debris from falling into the wells. Allow to incubate for at least 48 h.
	<ol>
		<li>Confirm the wells are completely coated via visual inspection. Ensure the elastomer is fully cured by gently prodding with a sterile pipette tip before removing.</li>
	</ol>
	</li>
	<li>Add 300 &#181;L of 70% ethanol (made with absolute ethanol and endotoxin-free water) and incubate at RT for 1 h. Remove the ethanol and perform endotoxin-free water washes by adding 300 &#181;L of endotoxin-free water to each well and incubating for 1 h (three times), 12 h, and 24 h prior to use to remove any remaining solvent.
	<ol>
		<li>Incubate 200 &#181;L of endotoxin-free water in three wells of each plate for 1 h. Measure endotoxin concentration of the water extracts using an endpoint chromogenic endotoxin assay (steps 2.5.1&#8722;2.5.6).</li>
	</ol>
	</li>
</ol>
4. Coating Cell Culture Surfaces with Fluorinated Poly(tetrafluoroethylene)
<ol>
	<li>Make a 1 mg/mL solution of fluorinated poly(tetrafluoroethylene) (fPTFE) (e.g., add 10 mg of fPTFE to 10 mL of fluorinated solvent [Table of Materials]) in a 20 mL glass scintillation vial. Place a magnetic stir bar in the vial and allow to stir for at least 24 h, until all solids are dissolved.</li>
	<li>Add approximately 150 &#956;L of the polymer solution to each well of a polystyrene 48-well plate (i.e., not tissue culture treated). Tilt the well plate slowly to ensure complete coverage of all wells with polymer solution. Replace lid.
	<ol>
		<li>To ensure effective fPTFE-coating of wells, glass coverslips should be coated in fPTFE and used for water contact angle measurement (step 4.3.1). Place coverslips inside the wells of a 24-well plate. Add approximately 400 &#956;L of the polymer solution to each well containing a coverslip. Push the coverslips down using sterile forceps, ensuring they are completely covered in polymer solution, and cover the well plate with a lid.</li>
	</ol>
	</li>
	<li>Place the well plate with polymer solution and/or coverslips into a vacuum oven set at 50 cmHg, 40 &#176;C. Remove the lid and cover with a single-ply wipe to prevent other debris from falling into the wells. Allow to incubate for at least 48 h.
	<ol>
		<li>Measure water contact angle of fPTFE-coated coverslips with a goniometer to ensure effective coating. 
		NOTE: Only water of the highest purity (e.g., glass triple distilled) should be used for water contact angle measurements.</li>
	</ol>
	</li>
	<li>Add 300 &#181;L of 70% ethanol (made with absolute ethanol and endotoxin-free water) and incubate at RT for 1 h. Remove the ethanol and perform endotoxin-free water washes by adding 300 &#181;L of endotoxin-free water to each well and incubating for 1 h (three times), 12 h, and 24 h prior to use to remove any remaining solvent.
	<ol>
		<li>Incubate 200 &#181;L of endotoxin-free water in three wells of each plate for 1 h. Measure endotoxin concentration of water extracts using an endpoint chromogenic endotoxin assay (steps 2.5.1&#8722;2.5.6).</li>
	</ol>
	</li>
	<li>UV sterilize the well plates for 30 min prior to cell culture experiments.</li>
</ol>
5. Making Lysate from 3T3 Cells
<ol>
	<li>Grow 3T3 cells in multiple T150 flasks to 70% confluence. To detach cells, aspirate media, wash surface with 5 mL of PBS, and aspirate PBS. Add 5 mL of animal origin-free, recombinant cell dissociation enzyme (Table of Materials) and incubate at 37 &#176;C for 3&#8722;5 min.</li>
	<li>Detach cells by gently tilting the flask back and forth. Add 5 mL of PBS to neutralize the recombinant enzyme used for cell dissociation. Transfer the detached cells from the flasks into a centrifuge tube and mix via pipetting. Perform a live cell count using a hemocytometer and cell viability dye. 
	NOTE: A cell dissociation enzyme that can be neutralized through dilution in PBS was selected to avoid the introduction of serum-based proteins in the lysate preparation. If trypsin is used to dissociate cells, it should be neutralized with a serum-containing solution, and an additional PBS wash should be performed to reduce the amount of serum proteins carried over into the lysate preparation.</li>
	<li>Centrifuge the cells at 200 x g for 5 min. Aspirate the supernatant and resuspend cells in original volume (i.e., 10 mL x number of flasks) of PBS to wash off any remaining media. Repeat.</li>
	<li>Centrifuge the cells again at 200 x g for 5 min and aspirate the supernatant. Add the volume of PBS required to achieve a final cell concentration of 1 x 106 cells/mL. Place the cell solution into a -80 &#176;C freezer until sample is fully frozen (at least 2 h).</li>
	<li>Thaw cell solution in a 37 &#176;C water bath. Once completely thawed, place the solution back into the -80 &#176;C freezer until totally frozen. Repeat for a total of 3 freeze-thaw cycles.</li>
	<li>Perform a micro bicinchoninic acid (BCA) assay on the cell lysate at a variety of dilutions (e.g., 1/100, 1/200, 1/500, 1/1000) to determine the protein concentration. Dilute the cell lysate to a protein concentration of 468.75 &#181;g/mL, aliquot, and store at -80 &#176;C for future use. 
	NOTE: Final protein concentration in a 48-well plate is 125 &#181;g/cm2 (based on the surface area of one well, 0.75 cm2).</li>
	<li>Perform a Western blot to assess presence of DAMPs in lysate (e.g., heat shock protein 60 [HSP60], high mobility group box 1 [HMGB1]) by loading 40&#8722;60 &#181;g of lysate protein in loading buffer onto a 1.5 mm thick 10% polyacrylamide gel and follow standard Western blot procedures.</li>
</ol>
6. Assessing Effect of Adsorbed Protein Layers and Toll-like Receptors on NF-&#954;B Activity of Macrophages
NOTE: For a schematic of the experimental workflow and plate layout, refer to Figure 1A and Supplemental Figure 1, respectively.
<ol>
	<li>Grow reporter macrophages in an appropriately sized flask to 70% confluence. Aspirate media, wash surface with PBS, and aspirate PBS. Add the recombinant cell dissociation enzyme and incubate at 37 &#176;C for 8 min.</li>
	<li>Detach cells by firmly tapping the sides of the flask. Inactivate the recombinant cell dissociation enzyme by adding an equal volume of growth media (containing 10% FBS). Perform a live cell count using a hemocytometer and cell viability dye. 
	NOTE: Expected viability for the reporter macrophages following an 8 min incubation in the cell dissociation enzyme is 90%.</li>
	<li>Centrifuge cells at 200 x g for 5 min. Aspirate supernatant and resuspend in original volume of PBS to wash cells. Centrifuge again and resuspend cells at 7.3 x 105 cells/mL in assay media (containing heat inactivated FBS).</li>
	<li>Separate cell suspension into 3 different tubes: TLR4 inhibitor, anti-TLR2, and untreated. Incubate cells with 1 &#181;g/mL TLR4 inhibitor for 60 min at RT or with 50 &#181;g/mL anti-TLR2 for 30 min at RT.</li>
	<li>Add 200 &#181;L of lysate, 10% FBS, 10% commercial mouse plasma (Table of Materials), or a mixture of the protein solutions to a 48-well plate (or equivalent) and allow protein to adsorb at 37 &#176;C for the desired amount of time (i.e., 30 min, 60 min, or 24 h). Aspirate protein solutions from wells, using a fresh Pasteur pipette for each protein solution, and wash surfaces with 250 &#181;L of PBS for 5 min. Aspirate PBS. Repeat for a total of 3 washes. 
	NOTE: This step may need to be started earlier in the protocol depending on desired adsorption time. Adjust protocol accordingly.</li>
	<li>After incubation period with the TLR4 inhibitor or anti-TLR2, pipette cells to resuspend. Add 200 &#181;L of cell solution to each well.</li>
	<li>For TLR2 positive control condition, add Pam3CSK4 to a final concentration of 150 ng/mL. For TLR4 positive control condition, add lipopolysaccharide (LPS) to a final concentration of 1.5 &#181;g/mL. Incubate cells at 37 &#176;C for 20 h.</li>
	<li>Sample 20 &#181;L of supernatant from each well and plate in duplicate into a 96-well plate. Include three wells of 20 &#181;L assay media as a background control. Add 200 &#181;L of SEAP reporter assay reagent to each well. Cover the plate with an adhesive seal and incubate for 2.5 h at 37 &#176;C. 
	NOTE: The incubation time may vary depending on experimental conditions, and should be optimized for a strong difference in absorbance between positive and negative control wells.
	<ol>
		<li>Transfer the remainder of the supernatant to a 1.5 mL tube (per well). Centrifuge at 1,000 x g for 10 min to pellet any debris. Transfer supernatant to a new 1.5 mL tube and store at -80 &#176;C. Analyze supernatant for the presence of proinflammatory cytokines (e.g., TNF-&#945;, interleukin 6) via enzyme-linked immunosorbent assay (ELISA).</li>
	</ol>
	</li>
	<li>Remove adhesive plate seal. Read absorbance of the plate using a plate reader at 635 nm. Aspirate liquid and discard plate.</li>
</ol>

The authors have nothing to disclose.

A primary focus of our lab is the host response to solid biomaterial soft tissue implants, and in particular how the cellular damage incurred during the implantation procedure impacts the host response. The work presented here describes preliminary experiments using a reporter macrophage cell line and in vitro-generated DAMP-containing cellular lysate, to investigate the influence of molecules released during cellular damage (i.e., from the implant surgery) on macrophage responses to biomaterials. Fibroblast cell lysate ...

The foreign body reaction (FBR) is a chronic host response that can negatively impact the performance of an implanted material or device (e.g., drug delivery devices, biosensors), through the persistent release of inflammatory mediators and by impeding integration between the implanted material and the surrounding tissue1. This innate immune response is initiated by the implantation procedure and is characterized by the long-term presence of innate immune cells and fibrous capsule formation around the implant1. Within the context of material host responses, macrophage-material interactions have a significant impact on the progression of the host response and development of a FBR1. Macrophages are a diverse innate immune cell population, recruited to the implant site either from tissue-resident macrophage populations or from the blood as monocyte-derived macrophages. They begin to accumulate at the implant site shortly after implantation, and within days become the predominant cell population in the implant microenvironment. Material-adherent macrophages, along with foreign body giant cells (FBGC) formed through macrophage fusion, can persist at the material surface for the lifetime of the implant2,3. Consequently, macrophages are considered to be key players in the foreign body response due to their roles orchestrating the characteristic steps of the FBR: acute inflammatory response, tissue remodeling, and formation of fibrotic tissue1.
Toll-like receptors (TLRs) are a family of pattern recognition receptors that are expressed by many immune cells, including macrophages, and have been shown to play a significant role in inflammation and wound healing. In addition to pathogen-derived ligands, TLRs are able to bind endogenous molecules, known as damage-associated molecular patterns (DAMPs), which are released during cell necrosis and activate inflammatory signaling pathways resulting in the production of proinflammatory cytokines4. We and others have proposed that damage incurred during soft tissue biomaterial implantation procedures release DAMPs, which then adsorb to biomaterial surfaces in addition to blood proteins and modulate subsequent cell-material interactions5,6. When macrophages interact with the adsorbed protein layer on an implant, their surface TLRs may recognize adsorbed DAMPs and activate proinflammatory signaling cascades, leading to NF-&#954;B and AP-1 transcription factor activation and production of proinflammatory cytokines. We have previously shown that murine macrophages have significantly increased NF-&#954;B/AP-1 activity and tumor necrosis factor &#945; (TNF-&#945;, proinflammatory cytokine) secretion in response to DAMP-containing adsorbed protein layers on a variety of polymeric surfaces compared to surfaces with adsorbed serum or plasma only (i.e., no DAMPs present), and that this response is largely mediated by TLR2, while TLR4 plays a lesser role5.
The NF-&#954;B/AP-1 reporter macrophage cell line (Table of Materials) used in this protocol is a convenient method to measure relative NF-&#954;B and AP-1 activity in macrophages5,7,8. In combination with TLR pathway inhibitors, this cell line is a useful tool for investigating TLR activation and its role in inflammation in response to a variety of stimuli5,7,8. The reporter cells are a modified mouse macrophage-like cell line that can stably produce secreted embryonic alkaline phosphatase (SEAP) upon NF-&#954;B and AP-1 transcription factor activation9. The colorimetric enzymatic alkaline phosphatase assay (Table of Materials) can then be used to quantify relative amounts of SEAP expression as an indirect measure of NF-&#954;B/AP-1 activity. As NF-&#954;B and AP-1 are downstream of many cell signaling pathways, neutralizing antibodies and inhibitors targeting specific TLRs (e.g., TLR2) or TLR adaptor molecules (e.g., MyD88) can be used to verify the role of a specific pathway. The methodology described in this article provides a simple and rapid approach for assessing the contribution of TLR signaling in murine macrophage responses to a variety of polymeric surfaces with adsorbed protein layers containing both blood proteins and DAMPs as an in vitro model of implanted biomaterials.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Cell culture reagents&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse/human CD282 (TLR2)</td><td>Biolegend</td><td>121802</td><td></td></tr><tr><td>CLI-095 (TLR4 inhibitor)</td><td>Invivogen</td><td>TLRL-CLI95</td><td></td></tr><tr><td>C57 complement plasma K2 EDTA 10ml, innovative grade US origin</td><td>InnovativeResearch</td><td>IGMSC57-K2 EDTA-Compl-10ml</td><td>Mouse plasma</td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Sigma Aldrich</td><td>D6429-500ML</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Fisher Scientific</td><td>14190250</td><td>No calcium, no magnesium</td></tr><tr><td>Fetal bovine serum (FBS), research grade</td><td>Wisent</td><td>98150</td><td></td></tr><tr><td>LPS-EK</td><td>Invivogen</td><td>TLRL-EKLPS</td><td>Lipopolysaccharide from Escherichia coli K12</td></tr><tr><td>NIH/3T3 fibroblasts</td><td>ATCC</td><td>CRL-1658</td><td></td></tr><tr><td>Pam3CSK4</td><td>Invivogen</td><td>tlrl-pms</td><td>Synthetic triacylated lipopeptide - TLR1/2 ligand</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>Plasmocin</td><td>Invivogen</td><td>ANT-MPP</td><td>Mycoplasma elimination reagent</td></tr><tr><td>RAW-Blue cells</td><td>Invivogen</td><td>raw-sp</td><td>NF-&amp;kappa;B/AP-1 reporter macrophage cell line</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>TrypLE express enzyme (1X)</td><td>Fisher Scientific</td><td>12604021</td><td>animal origin-free recombinant cell dissociation enzyme</td></tr><tr><td>Zeocin</td><td>Invivogen</td><td>ANT-ZN-1</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Kits and assays&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ELISA precoated plates, mouse IL-6</td><td>Biolegend</td><td>B213022</td><td></td></tr><tr><td>ELISA precoated plates, mouse TNF-&amp;alpha;</td><td>Biolegend</td><td>B220233</td><td></td></tr><tr><td>Endotoxin (Escherichia coli) - Control standard endotoxin (CSE)</td><td>Associates of Cape Cope Inc.</td><td>E0005-5</td><td>Endotoxin for standard curve in chromogenic endotoxin assay</td></tr><tr><td>LAL water, 100 mL</td><td>Associates of Cape Cope Inc.</td><td>WP1001</td><td>Used with chromogenic endotoxin assay</td></tr><tr><td>Micro BCA protein assay</td><td>Fisher Scientific</td><td>PI23235</td><td></td></tr><tr><td>Limulus amebocyte lysate (LAL) Pyrochrome endotoxin test kit</td><td>Associates of Cape Cope Inc.</td><td>C1500-5</td><td>Chromogenic endotoxin assay reagent</td></tr><tr><td>QUANTI-Blue alkaline phosphatase detection medium</td><td>Invivogen</td><td>rep-qb2</td><td>Alkaline phosphatase assay to indirectly measure NF-&amp;kappa;B/AP-1 activity</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Polymeric coating reagents&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chloroform, anhydrous</td><td>Sigma Aldrich</td><td>288306-1L</td><td></td></tr><tr><td>Ethyl alcohol anhydrous</td><td>Commercial Alcohols</td><td>P006EAAN</td><td>Sigma: Reagent alcohol, anhydrous, 676829-1L</td></tr><tr><td>Straight tapered fine tip forceps</td><td>Fisher Scientific</td><td>16-100-113</td><td></td></tr><tr><td>Fluorinert FC-40 solvent</td><td>Sigma Aldrich</td><td>F9755-100ML</td><td>Fluorinated solvent for fPTFE</td></tr><tr><td>Cell culture grade water (endotoxin-free)</td><td>Fisher Scientific</td><td>SH30529LS</td><td></td></tr><tr><td>Poly(methyl methacrylate) (PMMA)</td><td>Sigma Aldrich</td><td>182230-25G</td><td></td></tr><tr><td>Sylgard 184 elastomer kit</td><td>Fisher Scientific</td><td>50822180</td><td></td></tr><tr><td>Teflon-AF (fPTFE)</td><td>Sigma Aldrich</td><td>469610-1G</td><td>Poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene]</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Consumables&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adhesive plate seals</td><td>Fisher Scientific</td><td>AB-0580</td><td></td></tr><tr><td>Axygen microtubes, 1.5 mL</td><td>Fisher Scientific</td><td>14-222-155</td><td></td></tr><tr><td>Borosilicate glass scintillation vials, with white polypropylene caps</td><td>Fisher Scientific</td><td>03-337-4</td><td></td></tr><tr><td>Clear PS 48-well plate</td><td>Fisher Scientific</td><td>08-772-52</td><td></td></tr><tr><td>Clear TCPS 96-well plate</td><td>Fisher Scientific</td><td>08-772-2C</td><td></td></tr><tr><td>Clear TCPS 48-well plate</td><td>Fisher Scientific</td><td>08-772-1C</td><td></td></tr><tr><td>Cover glasses, circles</td><td>Fisher Scientific</td><td>12-545-81</td><td></td></tr><tr><td>Falcon tissue culture treated flasks, T25</td><td>Fisher Scientific</td><td>10-126-10</td><td></td></tr><tr><td>sticky-Slide 8 Well</td><td>Ibidi</td><td>80828</td><td></td></tr><tr><td>Superfrost microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Tissue culture treated flasks, T150</td><td>Fisher Scientific</td><td>08-772-48</td><td></td></tr></tbody></table>

a macrophage reporter cell assay to examine toll-like receptor-mediated nf-kb/ap-1 signaling on adsorbed protein layers on polymeric surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

Cleaning methods for the polymer-coated surfaces were tested to ensure there was no disruption of the coating, which would be seen as a change in the water contact angle to an uncoated glass coverslip (Figure 2). Soaking PMMA-coated microscope slides in 70% ethanol for 1 h was found to remove the PMMA coating (Figure 2, left panel), likely due to the solubility of PMMA in 80 wt% ethanol13, therefore PMMA-coated surfaces were cleaned using...

Watch this Scientific Journal Video about A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces at JoVE.com

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

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7.3K Views.  Queen's University. This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-кB/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

Video: A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Immunology and Infection

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biology

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicine

Detection of Inflammasome Activation via Fluorescence Microscopy

Source: den Hartigh, A. B. et al., <a href="https://app.jove.com/v/57463">Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.</a> J. Vis. Exp.&#160;(2018)
This video demonstrates a method to detect NLRP3 inflammasome activation in macrophages using fluorescent reporter probes. The cells are treated with potassium ionophores and glycine to induce inflammasome assembly and caspase-1 activation. Fluorescent reporter probes visualize activated caspase-1, and cells are stained with a fluorescent dye for nuclei visualization.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

Fluorescence Based Transwell Macrophage Attraction Assay: An In Vitro Method to Study Tumor-Macrophage Interaction Through Chemical-Induced Chemotaxis

Source: An, Z. et al. <a href="https://www.jove.com/v/59907/in-vitro-assay-to-study-tumor-macrophage-interaction">In Vitro Assay to Study Tumor-macrophage Interaction.</a> J. Vis. Exp. (2019)
In this video, we study tumor-macrophage interaction using&#160;fluorescence-based transwell macrophage interaction assay. The assay involves the analysis of the migration of macrophages through chemical-induced chemotaxis.

Source: An, Z. et al. In Vitro Assay to Study Tumor-macrophage Interaction. J. Vis. Exp. (2019)
In this video, we study tumor-macrophage interaction ...

Cancer Research

Cancers of the Nervous System

In vitro studies

An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition

Source: Yang, H., et al. <a href="https://app.jove.com/v/56075">Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling.</a> J. Vis. Exp. (2017).
This video demonstrates a reporter cell-based assay for screening potential bioactive nanoparticles that inhibit Toll-like receptor (TLR) signaling. The assay involves introducing lipopolysaccharides (LPS) mixed with peptide-gold nanoparticle hybrids to reporter macrophages expressing the reporter proteins &#8212; secreted embryonic alkaline phosphatase (SEAP) and luciferase. The reduction of LPS-induced TLR signal mediated by the hybrids is determined by measuring the reporter signals.

1. Screening for Potential TLR4 Nano-inhibitors Using the Reporter Cells
NOTE: Since TLR4 signaling utilizes both MyD88-dependent land TRIF-dependent ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Summary

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Chapters in this video

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Detection of Inflammasome Activation via Fluorescence Microscopy

Fluorescence Based Transwell Macrophage Attraction Assay: An In Vitro Method to Study Tumor-Macrophage Interaction Through Chemical-Induced Chemotaxis

An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition