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

Vizyon Sağlık Araştırma Ağı - Bu çalışma Sağlık Araştırması (178.918), Fonds de recherche en santé du Québec, Kanada Enstitüleri hibe ile desteklenmektedir.

Not: Erkek C57BL / 6 fareleri (5-7 haftalık), Charles River Laboratories International, Inc. (. St-Constant, Quebec, Kanada) ve MUB Sainte-Justine Hayvan Bakım Komitesi tarafından onaylandı protokollere göre manipüle edilebilir. Fare bronş doku eksplantlarında epitelial hücreler üzerinde LMPs proapoptotik etkilerinin araştırılması için primer bronşiyal epitel hücreleri için iyi bir kaynak sağlamaktadır. Bu protokol LMPs in vitro üretimi ve aynı zamanda LMPs ile muamele edilmiş bronş doku...

<ol>
	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. 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. 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=Sickle+blood+contains+tissue+factor-positive+microparticles+derived+from+endothelial+cells+and+monocytes.">Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes.</a> Blood. 102 (7), 2678-2683 (2003).</li><li>Mause, S. F., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+protagonists+of+a+novel+communication+network+for+intercellular+information+exchange.">Microparticles: protagonists of a novel communication network for intercellular information exchange.</a> Circ Res. 107 (9), 1047-1057 (2010).</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=Anti-proliferative+and+anti-tumour+effects+of+lymphocyte-derived+microparticles+are+neither+species-+nor+tumour-type+specific.">Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific.</a> J Extracell Vesicles. 3, (2014).</li><li>Soleti, R., 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=Microparticles+harboring+Sonic+Hedgehog+promote+angiogenesis+through+the+upregulation+of+adhesion+proteins+and+proangiogenic+factors.">Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors.</a> Carcinogenesis. 30 (4), 580-588 (2009).</li></ol>

Milletvekilleri hücrelerarası çapraz konuşma aktif arabulucular ve onların çalışma bilimin birçok alanda umut vericidir. 11 Bu çalışma bir apoptotik T hücre soyu elde LMPs in vitro büyük ölçekli üretimi için ayrıntılı bir protokol sundu. Bu MPler lenfosit moleküllerinin büyük bir repertuarı ifade eder ve biyolojik hücre ve doku homeostazının düzenlenmesinde rol oynar. Bununla birlikte, farklı kaynaklardan elde edilen LMPs biyolojik olarak farklı olabilir. 4,9,12,1...

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 height="21" style="height:21px;width:303px;">LMPs production and characterization</td>
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			<td>BD Bio-sciences</td>
			<td style="width:124px;">FACS Calibur</td>
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			<td height="21" style="height:21px;width:303px;">Spectrophotometer</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Series 600</td>
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			<td height="21" style="height:21px;">Bronchial tissue explants and sections&#160;</td>
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			<td style="width:231px;">Becton Dickinson and Company</td>
			<td style="width:124px;">353043</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

Hücre-hücre iletişimine hücre zarı türetilmiş veziküllerin biyolojik rolleri ilgi son yıllarda artmıştır. Mikropartiküller (MP) ile 1 um, 0.1 um çapı kadar, veziküllerin böyle bir tipi olup, tipik olarak aktivasyonu ya da apoptoza ökaryotik hücrelerin plazma zarından döküldü. Burada D LMPs çok aşamalı bir diferansiyel santrifüj işlemi ile izole edilir ve akış sitometrisi kullanılarak, özelliği aktinomisin ile uyarılan apoptotik CEM T hücreleri T lenfosit kaynaklı mikro (LMPs) üretimini tarif eder. Bu protokol, aynı zamanda, fare primer solunum bronş doku eksplantlarında türetilen bronş epitel hücrelerinde LMPs proapoptotik etkilerini kanıtlamak için in situ hücre ölümü algılama yöntemi sunulur. Burada tarif edilen yöntemler, in vitro apoptotik lenfositlerden LMPs, çok miktarda izole etmek için tekrarlanabilir bir yöntem sağlamaktır. LMPs türetilmişBu şekilde, çeşitli hastalık modellerinde özelliklerini değerlendirmek için kullanılır ve farmakoloji ve toksikoloji testleri için edilebilir. Solunum yolu epitel dış çevre ve altta uzanan doku arasındaki bir koruyucu fiziksel ve fonksiyonel bariyer sunar göz önüne alındığında, bronşiyal doku eksplantlarında yerine ölümsüz epitel hücre çizgilerinin kullanımı yolu sistemi doku gerektiren araştırmalar için etkili bir model sağlar.

LMPs floresan-aktive edilmiş hücre (FACS) analizi, sıralama ve MP&#39;lerin (≤1 um)% 97 anneksin-V-Cy5 pozitif olan 1 um boncuklar kullanılarak bir kapı ile anneksin V boyama 10 ile karakterize edilmiştir (Şekil 1A ve 1B). Tipik haliyle, LMPs yaklaşık 2.5 mg ile bu protokol izlenerek elde edilmiştir. C57BL bronş doku eksplantlarında / 6 fareleri araç ve LMPs işlemine tabi tutulmuştur. Bronşiyal bölümlerin histopatolojik analizi bronşiyal epitel yapısal bütünlüğü ile...

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 (Video) | JoVE

Hücre zarı atkı mikroparçacıklar (MPler), izole edilmiş ve kendi patofizyolojik etkileri, çeşitli modelleri üzerinde araştırılabilir aktif biyolojik veziküllerdir. Burada T lenfositleri (LMPs) türetilen milletvekillerini üretilmesi için ve solunum yolu epitel hücreleri üzerindeki proapoptotik etkilerini kanıtlamak için bir yöntem tarif eder.

Havayolu Epitel Hücreleri üzerinde Lenfositik mikrotanecikler ve proapoptotik Etkisi Algılama Üretimi

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

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

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 (Video) | JoVE

Co-kültür Modelleri Pseudomonas aeruginosa</emCanlı İnsan Havayolu Hücreler Yetiştirilen> Biyofilmler

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce (Video) | JoVE

Yapışkan bant tabanlı Örnekleme ve Floresans kombinasyonu In situ</emHızlı Algılama> Hibridizasyon Salmonella Taze üretin

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 PeyerÃ¢â‚¬â„¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells (Video) | JoVE

Fare Uygulama M hücreleri tarafından Bakteriyel Alımı değerlendirin Peyer Patch Bağırsak Loop Testi bağlandı

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

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 (Video) | JoVE

Polarize İnsan Havayolu bir sıvı kaplı Kültürünün Oluşturulması Solunum Patojenler için Host Hücre Yanıtı Okumak Epitel Calu-3 Hücreler<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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus (Video) | JoVE

İnsan Thymus ile myeloid dendritik hücreleri ve epitel hücreleri İzolasyonu

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

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells (Video) | JoVE

Kırmızı kan hücreleri üzerinde antikor kaynaklı kompleman aktivasyon sayısal tespiti için Yöntemler

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

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells (Video) | JoVE

Retina Pigment Epitel Hücreleri üzerinde Fagositoz Tahliller için Domuz veya Sığır fotoreseptör dış Kesimleri Büyük Ölçekli Arıtma

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

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

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice (Video) | JoVE

Fenotipik ve Kronik Lenfositik koryomenenjit Virüs ile enfekte Fare İzole Aktif Düzenleyici T hücrelerinin Fonksiyonel Analiz

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 (Video) | JoVE

Mukozal astar sıvı noninvaziv örnekleme için In Vivo üst solunum yolu bağışıklık arabulucu düzeyleri miktar

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

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies (Video) | JoVE

Yalıtım ve nötrofil kaynaklı Microparticles karakterizasyonu için fonksiyonel çalışmalar

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