eoe sub articles

EoE Sub Articles

1. Screening for Potential TLR4 Nano-inhibitors Using the Reporter Cells
NOTE: Since TLR4 signaling utilizes both MyD88-dependent land TRIF-dependent pathways, it is selected as the primary target to encompass a wide range of TLR signaling pathways. THP-1-XBlue reporter cells are used to mainly examine the NF-&#954;B/AP-1 activation while THP-1-Dual cells are for IRF activation from the TRIF-dependent signal transduction.
<ol>
	<li>Identify the optimal LPS dose by generating a dose-response curve prior to the screening.&#8203;
	<ol>
		<li>Dilute the working LPS solution to a final concentration of 0.01 - 100 ng/mL in R10 medium (make dilution in log10 scale). Layout the plate design and make dilution in a 96-well round-bottom culture plate. Make sure that each well has a minimum of 110 &#956;L of solution after dilution.</li>
		<li>Gently remove the culture medium from the cell-seeded plate using a vacuum aspirator.</li>
		<li>Transfer the LPS containing R10 medium prepared in the dilution plate (1.1.1) into the culture plate according to the layout of the samples. Incubate the plate at 37 &#176;C (in a cell culture incubator) for 24 h.</li>
		<li>At 24 h, carefully transfer the supernatants (80 &#956;L/well) into a new 96-well round-bottom plate. Conduct the colorimetric and/or luciferase luminescence assay on these solutions immediately or store them at 4 &#176;C (several hours) or -20 &#176;C (days) prior to the assay development. 
		NOTE: The design of the plate layout should be simple and clear for experiment and data analysis. For each condition, 2-4 replicates should be considered. Always include a negative control group (LPS null). It is recommended to use THP-1-XBlue cells for NF-&#954;B/AP-1 activation as the Dual cells have a relatively high background of NF-&#954;B/AP-1 activation after being differentiated into macrophages. However, it is feasible to use the Dual cells for reporting both NF-&#954;B/AP-1 and IRF activation.</li>
	</ol>
	</li>
	<li>Develop the colorimetric assay for the NF-&#954;B/AP-1 activation and the luciferase luminescence assay for IRF activation.
	<ol>
		<li>To assess the NF-&#954;B/AP-1 activation, transfer 20 &#956;L of supernatant of each sample into a new 96-well flat-bottom culture plate, and add 180 &#956;L of pre-warmed SEAP substrate solution into each well. Incubate the plate at 37 &#176;C for 1 - 2 h to allow the color development (pink to dark blue). Collect the absorption at 655 nm on a plate reader. 
		CAUTION: The incubation time can be varied based on the color development (30 min up to overnight incubation). It is recommended to wait until the optical density (O.D.) of the darkest color reaches above 1 while avoiding the saturation of the color development (O.D &#62;3).</li>
		<li>For the analysis of IRF activation, transfer 10 &#956;L of supernatant of each sample into a 96-well clear flat-bottom white plate. Add the luciferase solution (50 &#956;L) and immediately collect the luminescence well by well. 
		NOTE: It is highly recommended to use a luminescence plate reader with an auto-injection function to ensure consistency in the luminescence reading from well to well and from plate to plate. If the substrate solutions are manually injected, please ensure a consistent incubation time and reading set-up for each well.</li>
	</ol>
	</li>
	<li>Conduct the screening assay on various peptide-GNP hybrids with 10 ng/mL LPS stimulation (based on 1.1). Follow the same procedure in 1.2 for the reporter assay development.
	<ol>
		<li>Concentrate the peptide-GNP hybrids to 200 nM in an R10 medium using the centrifugation method. Centrifuge 20 volumes of the hybrid solution (10 nM) at 18,000 x g for 30 min, and carefully discard the supernatants. Collect the hybrids (at the bottom of the tube) into a tube, wash them with PBS twice, and re-suspend the hybrids in one volume of the R10 medium.</li>
		<li>Mix equal volumes of the concentrated hybrids and the LPS (20 ng/mL) containing R10 medium to have a final concentration of the hybrids and LPS to be 100 nM and 10 ng/mL, respectively.</li>
		<li>Remove the culture medium from the cultured plate and add 100 &#956;L of the mixed solution into each well (3 replicates for each condition); include a negative control (medium only) and a LPS control (10 ng/mL LPS without hybrids).</li>
		<li>After 24 h incubation at 37 &#176;C, transfer the medium of each well into a tube and centrifuge the tubes at 18,000 x g, 4 &#176;C for 30 min. Collect the supernatants (50-80 &#956;L/tube) into a 96-well round-bottom plate, and perform the reporter assay as described in 3.2. 
		CAUTION: When discarding the supernatants after centrifugation, do not agitate the hybrids at the bottom of the tube. 
		NOTE: The centrifugation in step 1.3.4 is very important to remove the non-internalized nanoparticle hybrids from the culture medium, and can avoid the interference of the hybrids with the colorimetric/luminescence readings. This is because the gold nanoparticles can absorb wide wavelengths of light depending on their size and agglomeration.</li>
	</ol>
	</li>
</ol>
2. Validating the Inhibitory Effect of the Potential Candidates
NOTE: To confirm the inhibitory effect of the potential candidates from the screening, two approaches are employed. One is to examine the dose responses of the stimulants (LPS) at a fixed hybrid concentration (or the other way around); the other is to directly look at the inhibition of the NF-&#954;B/AP-1 and IRF3 signals via immunoblotting.
<ol>
	<li>In approach one, perform the reporter assay with 100 nM of the lead hybrids and two LPS concentrations at 1 ng/mL and 10 ng/mL following the same procedures described in 1.3. Include an inactive hybrid (based on the screening results) as a hybrid control for comparison.</li>
	<li>For the immunoblotting approach, please follow the standard experimental procedures.
	<ol>
		<li>Culture THP-1 cells in R10 medium, seed the cells into a 12-well culture plate (2 x 106 cells/well), and differentiate them into macrophages by treating the cells with 50 ng/mL PMA for 24 h followed by resting for 2 days.</li>
		<li>After cell differentiation, stimulate cells with 10 ng/mL LPS with/without the hybrids (100 nM) over time (up to 4 h). At various time points (0, 5, 15, 30, 60, 120, and 240 min), prepare the cell lysates for immunoblotting. Include an inactive hybrid as a control.</li>
		<li>Probe the signals of I&#954;B&#945;, phosphorylated p65, and phosphorylated IRF3 to examine the inhibitory effect of the lead hybrids on the activation of NF-&#954;B and IRF3. Probe the &#946;-actin and total IRF3 signals as internal controls. 
		NOTE: The signal transduction often occurs much faster (in a few hours) than the expression of the reporter enzymes SEAP and luciferase (24 h). It is highly recommended to also perform a viability assay of the tested hybrids at 24 h as another validation method.</li>
	</ol>
	</li>
</ol>
3. Evaluating the TLR Specificity
NOTE: To investigate the TLR specificity of the lead peptide-GNP hybrid, other TLR signaling pathways are tested, including TLR2, TLR3, and TLR5. TLR7, 8, and 9 are excluded because the THP-1-derived macrophages do not respond well to the stimulation of these TLRs due to the lack of TLR7, 8, and 9 expression in macrophages.
<ol>
	<li>Test various concentrations (1 ng/mL to 25 &#956;g/mL) of the ligands specific to TLR2 (Pam3CSK4), TLR3 (poly I:C), and TLR5 (flagellin) on both reporter cells derived macrophages to obtain the optimal concentration following the same procedure in 1.1 and 1.2.</li>
	<li>Treat the cells with the mixtures of the lead hybrid (100 nM) and each TLR ligand at the concentration obtained from 3.1 to evaluate the inhibitory specificity of the lead hybrid according to the experimental procedure described in 1.3. Include the inactive hybrid as a control for comparison.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>THP-1-XBlue reporter cell</td>
			<td>InvivoGen</td>
			<td>thpx-sp</td>
			<td>keep cell culture passage under 20</td>
		</tr>
		<tr>
			<td>THP-1-Dual repoter cell</td>
			<td>InvivoGen</td>
			<td>thpd-nfis</td>
			<td>keep cell culture passage under 20</td>
		</tr>
		<tr>
			<td>RPMI-1640 (no L-glutamine)</td>
			<td>GE Health Care</td>
			<td>SH30096.02</td>
			<td>Warm up to 37 &#176;C before use; add supplements to make a complete medium R10</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (qualified)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12484028</td>
			<td>Heat inactivated; 10% in RPMI-1640</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30034.02</td>
			<td>2 mM in the complete medium R10</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td>1 mM in the complete medium R10</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, 1X, without calcium, magnesium</td>
			<td>GE Health Care</td>
			<td>SH30028.02</td>
			<td>Use for cell washing and reagent preparation</td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>InvivoGen</td>
			<td>rep-qb1</td>
			<td>SEAP substrate</td>
		</tr>
		<tr>
			<td>QUANTI-Luc</td>
			<td>InvivoGen</td>
			<td>rep-qlc2</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>InvivoGen</td>
			<td>ant-zn-1</td>
			<td>Selection antibiotics for reporter cells</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>InvivoGen</td>
			<td>anti-bl-1</td>
			<td>Selection antibiotics for reporter cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO) for molecular biology</td>
			<td>Sigmal-Aldrich</td>
			<td>D8418-100ML</td>
			<td>Use for reagent preparation</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS) from E. coli K12</td>
			<td>InvivoGen</td>
			<td>tlrl-eklps</td>
			<td>TLR4 ligand</td>
		</tr>
		<tr>
			<td>Flagellin from S. Typhimurium (FLA-ST), ultrapure</td>
			<td>InvivoGen</td>
			<td>tlrl-epstfla</td>
			<td>TLR5 ligand</td>
		</tr>
		<tr>
			<td>SpectraMax Plus 384 microplate reader</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Read colorimetric assay</td>
		</tr>
		<tr>
			<td>Infinite M200 Pro multimode microplate reader with injectors</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Read luminiscience</td>
		</tr>
		<tr>
			<td>Costar assay plate, 96-well white with clear flat bottom, tissue culure treated</td>
			<td>Corning Costar</td>
			<td>3903</td>
			<td>Used for luminiscence assay</td>
		</tr>
	</tbody>
</table>

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.

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

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

Take a multiwell plate with different reporter macrophages harboring the secreted embryonic alkaline phosphatase, or SEAP, gene or a secreted luciferase gene under inducible promoters.
Add media containing different peptide-gold nanoparticle hybrids mixed with lipopolysaccharides or LPS to designated wells.
LPS binds to macrophage toll-like receptors, or TLR, triggering internalization into the endosome.
The endosomal proton pump transports protons inside. The acidification triggers TLR signaling, activating transcription factors.
These factors induce the expression of the reporter genes, producing SEAP or luciferase.
Alternatively, internalization of LPS along with the hybrids causes the negatively charged peptide groups to sequester luminal protons, preventing endosomal acidification and inhibiting TLR signaling.
Collect the media, centrifuge to separate the supernatant, and add substrates for SEAP and luciferase.
SEAP converts its substrate to a colored product, while luciferase catalyzes its substrate to produce luminescence.
A reduction in reporter signals indicates the effectiveness of the hybrids in TLR signaling inhibition.

an-assay-to-screen-bioactive-nanoparticles-for-toll-like-receptor

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

79 Views. Source: Yang, H., et al. Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling. 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 — secreted embryonic alkaline ...

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

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

JoVE Journal

Bioengineering

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Immunology and Infection

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.

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 ...

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

Transcript

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

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

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