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EoE Sub Articles

1. Measurement of bioactive type-I IFN using HEK-Blue IFN-&#945;/&#946; reporter cells.
Note: These cells were generated by stable transfection of HEK293 cells with the human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. They also contain the secreted alkaline phosphatase (SEAP) reporter gene under the control of the IFN-&#945;/&#946; inducible ISG54 promoter. Stimulation of these cells with type-I IFN activates the JAK/STAT/ISGF3 pathway and induces the production of SEAP.
<ol>
	<li>Maintain HEK-Blue IFN-&#945;/&#946; cells in DMEM supplemented with 10% FBS. Plate them at a density of 50,000 cells per well in a flat-bottom 96-well plate, with a final volume of 180 &#181;l.</li>
	<li>Mix 220 &#181;l of PBMCs (diluted at 850,000 cells/ml) or 220 &#181;l of plasmacytoid dendritic cells (pDCs) (at a concentration ranging from 100,000 to 500,000 cells/ml) with 30 &#181;l of infected T cells (diluted at 106 cells/ml) per well in a U-bottom 96 well plate.</li>
	<li>Incubate co-cultures at 37 &#176;C and 5% CO2 for 18-22 hr, and then transfer them to a V-bottom 96 well plate. Centrifuge for 5 min at 400 x g.</li>
	<li>Add 20 &#181;l of co-culture supernatant to each well in duplicate. Each plate also requires a set of internal standard controls (human IFN&#945;, final concentration from 100 U/ml to 2,500 U/ml). Incubate the plates at 37 &#176;C and 5% CO2 for 18-22 hr.</li>
	<li>Determine levels of alkaline phosphatase activity using QUANTI-Blue solution, which changes from pink to purple/blue in the presence of the enzyme. Prepare QUANTI-Blue following the manufacturer&#39;s recommendations. Add 180 &#181;l of this solution to each well of a flat-bottom 96-well plate. To each well also add 20 &#181;l of the induced HEK-Blue IFN-&#945;/&#946; cells supernatants and incubate the plate in a 37 &#176;C incubator until color develops in the standard IFN control wells.</li>
	<li>Evaluate levels of SEAP using a spectrophotometer at 620-655 nm and determine the concentration of type-I IFN by extrapolation from the linear part of the IFN standard curve.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MT4 cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>120</td>
			<td>This reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: MT-4 from Dr. Douglas Richman.</td>
		</tr>
		<tr>
			<td>HEK-Blu IFN-&#945;/&#946; reporter cells</td>
			<td>Invivogen</td>
			<td>HKB-IFNAB</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Wisent</td>
			<td>350-000-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>PHA-L</td>
			<td>Sigma-Aldrich</td>
			<td>O2769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation kit II</td>
			<td>Myltenyi</td>
			<td>130-091-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Plasmacytoid Dendritic Cell Isolation Kit II</td>
			<td>Myltenyi</td>
			<td>130-097-240</td>
			<td></td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>Cedarlane</td>
			<td>REP-QB2</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mg/ml human IgG</td>
			<td>Sigma-Aldrich</td>
			<td>I 4506</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of bioactive type-i interferons using a secreted embryonic alkaline phosphatase reporter assay

Source: Bego, M. G. et al., <a href="https://app.jove.com/v/51207">Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.</a> J. Vis. Exp. (2015)
This video demonstrates secreted embryonic alkaline phosphatase assay using HEK-Blue interferon-&#945;/&#946; reporter cells to quantify the type-I interferons (IFNs) released following stimulation of plasmacytoid dendritic cells by HIV-1 Infected CD4+ T cells.

Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay

Source: Bego, M. G. et al., Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture ...

To quantify the bioactive type-I interferons, such as interferon-alpha and interferon-beta, in a test sample, take a multi-well plate containing engineered reporter cells. These cells express the secreted embryonic alkaline phosphatase, SEAP, reporter gene under the control of the interferon-alpha and beta inducible promoter.
Add the test sample containing an unknown type-I interferon concentration. Pipette a range of human type-I interferon concentrations to other wells, which act as standard controls. Incubate.
Type-I interferon in the test well binds to a specific cell surface receptor, activating the Janus kinases. These protein kinases phosphorylate signal transducers and activators of transcription, STAT, proteins, which dimerize and form a complex with a specific interferon regulatory factor.
Upon entering the nucleus, the complex binds to a specific DNA sequence in the interferon-alpha and beta inducible promoter, activating SEAP gene transcription and producing the SEAP enzymes, which are secreted into the media.
Transfer the SEAP-containing media to multi-well plate wells having a SEAP-specific substrate. SEAP hydrolyzes the pink substrate, producing a purple-blue-colored product.
Using a microplate reader, measure the product&#39;s absorbance in the test and standard wells, indicative of SEAP levels and Type-I interferon concentration.
From the plotted standard type-I interferon curve, determine the bioactive type-I interferon concentration in the test sample.

quantification-bioactive-type-i-interferons-using-secreted-embryonic

Research

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Immunology

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60 조회수. 출처: Bego, M. G. et al., Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-Culture System. J. Vis. 특급 (2015) 이 비디오는 HIV-1에 감염된 CD4+ T 세포에 의한 형질세포(plasmacytoid dendritic cell)의 자극 후 방출되는 I형 인터페론(IFN)을 정량화하기 위해 HEK-Blue 인터페론-α/β 리포터 세포를 사용하여 분비된 배아 알칼리 인산가수분해효소 분석을 보여줍니다. 

비디오: Secreted Embryonic Alkaline Phosphatase Reporter Assay를 사용한 Bioactive Type-I 인터페론의 정량화 - 개념

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

HIV-1 양성 개인의 플라즈마에서 엑소 좀의 분리

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

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JoVE Journal

면역학 및 감염병학

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

대사 표현형을 각기 다른 미성숙, 성숙 및 Tolerogenic 수지상 세포의 생성

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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.

고분자 표면에 흡착 된 단백질 층에 유료 수용체 매개 NF-kB / AP-1 신호를 검사하는 대식 세포 분석

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

Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay

내레이션 대본

Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay