Name: generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells
Uploaded: 2015-02-20T15:00:00
Description: Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Dit werk wordt ondersteund door subsidies van de Canadese Institutes of Health Research (178.918), Fonds de recherche en santé du Québec - Vision Health Research Network.

OPMERKING: Man C57BL / 6 muizen (5-7 weken oud) zijn van Charles River Laboratories International, Inc. (St-Constant, Quebec, Canada.) En gemanipuleerd volgens de protocollen door de CHU Sainte-Justine Animal Care Comité is goedgekeurd. Mouse bronchiale weefselexplantaten een goede bron van primaire bronchiale epitheelcellen onderzoeken van de pro-apoptotische effecten van LMPs op epitheelcellen. Dit protocol beschrijft de in vitro generatie van LMPs, alsmede een werkwijze voor het detecteren van apoptotische ...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. 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Kamerleden zijn actieve bemiddelaars van intercellulaire overspraak en hun studie is veelbelovend in veel gebieden van de wetenschap. 11 Deze studie presenteerde een gedetailleerd protocol voor in vitro grootschalige opwekking van LMPs afgeleid van een apoptotische T-cellijn. Deze Kamerleden drukken een groot repertoire van lymfocyten moleculen en zijn biologisch betrokken bij de regulatie van de cellulaire en weefselhomeostase. Echter, LMPs afkomstig van verschillende bronnen kunnen biologisch versc...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

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			<td style="width:124px;">Series 600</td>
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			<td style="width:171px;">Straight, sharp, 9cm longth</td>
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generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

De belangstelling voor de biologische rol van cel-membraan afgeleide blaasjes in cel-cel communicatie is de afgelopen jaren toegenomen. Microdeeltjes (MPs) zijn een dergelijk type van vesicles, variërend in diameter van 0,1 urn tot 1 urn, en kenmerkend schuur van het plasmamembraan van eukaryote cellen die activering of apoptose. Hier beschrijven we de generering van T-lymfocyten afkomstige microdeeltjes (LMPs) van apoptotische CEM T-cellen gestimuleerd met actinomycine D. LMPs worden geïsoleerd door een meerstaps proces differentiële centrifugatie en gekarakteriseerd met behulp van flowcytometrie. Het protocol stelt ook een in situ celdood detectiemethode voor aantonen van de pro-apoptotische effect van LMPs op bronchiale epitheelcellen afkomstig uit muizen primaire luchtwegen bronchiale weefselexplantaten. Hierin beschreven werkwijzen verschaffen een reproduceerbare werkwijze voor het isoleren overvloedige hoeveelheden LMPs van apoptotische lymfocyten in vitro. LMPs afgeleidop deze wijze kan worden gebruikt om de kenmerken van diverse ziektemodellen evalueren en farmacologie en toxicologie getest. Aangezien het luchtwegepitheel biedt een beschermende fysieke en functionele barrière tussen de buitenomgeving en het onderliggende weefsel, gebruik van bronchiale weefselexplantaten plaats geïmmortaliseerde epitheliale cellijnen een effectief model voor het onderzoek ter luchtwegen luchtwegweefsel.

LMPs werden gekenmerkt met annexine V-kleuring 10 door fluorescentie-geactiveerde cel sortering (FACS) analyse en gated met behulp van 1 micrometer kralen, waarin 97% van de parlementsleden (≤1 micrometer) waren annexine-V-Cy5 positief (figuur 1A en 1B). Typisch, ongeveer 2,5 mg LMPs werden verkregen na dit protocol. Bronchiale weefselexplantaten van C57BL / 6 muizen werden blootgesteld aan het voertuig en LMPs behandeling. Histopathologische analyse van bronchiale secties toonde het effect...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Celhuid werpen microdeeltjes (MPs) actief biologische blaasjes die kunnen worden geïsoleerd en de pathofysiologische effecten onderzocht in verschillende modellen. Hier beschrijven we een werkwijze voor het genereren MPs afgeleid van T-lymfocyten (LMPs) en voor het aantonen van hun pro-apoptotische effect op epitheelcellen.

Generatie Lymfatische micropartikels en Detectie van hun proapoptotische Effect op epitheelcellen

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Research

JoVE Journal

Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-cultuur modellen van Pseudomonas aeruginosa Biofilms Grown op Live menselijke luchtwegen cellen

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

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Combinatie van Adhesive-tape-gebaseerde Sampling en fluorescentie In situ Hybridisatie voor snelle detectie van Salmonella Op Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Toepassing van een muis geligateerd Peyer&#39;s Patch Intestinale Loop Assay om Bacteriële opname evalueren door M-cellen

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Het synergetische effect van zichtbaar licht en gentamycine op<em&gt; Pseudomona aeruginosa</em&gt; Micro-organismen

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Tot oprichting van een Liquid bedekte cultuur van gepolariseerde menselijke luchtwegen Epiteel Calu-3 cellen aan Host Cell Reactie op respiratoire pathogenen Studie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolatie van myeloïde dendritische cellen en epitheelcellen van Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Methoden voor de kwantitatieve detectie van antilichaam-geïnduceerde complement activatie op de rode bloedcellen

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Fenotypische en functionele analyse van geactiveerde regulatoire T-cellen Geïsoleerd van Chronische Lymfatische choriomeningitis-virus geïnfecteerde muizen

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Noninvasive bemonstering van Mucosal voering vloeistof voor de kwantificering van In Vivo bovenste luchtwegen immuun-bemiddelaar niveaus

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Isolatie en karakterisatie van neutrofiele afkomstige deeltjes voor functionele Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...