Name: a macrophage reporter cell assay to examine toll-like receptor-mediated nf-kb/ap-1 signaling on adsorbed protein layers on polymeric surfaces
Uploaded: 2020-01-07T12:00:00
Description: 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

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 f...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Cell culture reagents</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&#39;s modified eagle medium (DMEM)</td>
			<td>Sigma Aldrich</td>
			<td>D6429-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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-&#954;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 colspan="4">Kits and assays</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-&#945;</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-&#954;B/AP-1 activity</td>
		</tr>
		<tr>
			<td colspan="4">Polymeric coating reagents</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 colspan="4">Consumables</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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