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

Ce travail est soutenu par des subventions des Instituts de recherche en santé (178 918), le Fonds de recherche en santé du Québec du Canada - Réseau de recherche sur la santé visuelle.

REMARQUE: Homme souris C57BL / 6 (ancienne 5-7 semaines) sont de Charles River Laboratories International, Inc. (St-Constant, Québec, Canada.) Et manipulés selon des protocoles approuvés par la Sainte-Justine Comité de protection des animaux CHU. Souris explants de tissus bronchiques constituent une bonne source de cellules épithéliales bronchiques primaires pour étudier les effets pro-apoptotiques de PMT sur les cellules épithéliales. Ce protocole décrit la génération in vitro de PMT, ainsi qu&#39;...

<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>

Les députés sont des médiateurs actifs de diaphonie intercellulaire et leur étude est prometteur dans de nombreux domaines de la science. 11 Cette étude a présenté un protocole détaillé pour in vitro production à grande échelle de PMT issues d&#39;une lignée de cellules T apoptotique. Ces députés expriment un large répertoire de molécules de lymphocytes et sont biologiquement impliqués dans la régulation de l&#39;homéostasie cellulaire et tissulaire. Cependant, PMT provenant de dif...

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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	<tbody>
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			<td height="21" style="height:21px;width:303px;">LMPs production and characterization</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td style="width:171px;"></td>
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			<td height="21" style="height:21px;">CEM T cells&#160;</td>
			<td style="width:231px;">ATCC&#160;</td>
			<td style="width:124px;">CCL-119</td>
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			<td height="21" style="height:21px;width:303px;">X-VIVO 15 medium&#160;</td>
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			<td>04-744Q</td>
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			<td style="width:124px;">83.1812.502</td>
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			<td>14190-144</td>
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			<td style="width:124px;">83.1826.001</td>
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			<td>Falcon</td>
			<td style="width:124px;">352058</td>
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			<td height="21" style="height:21px;width:303px;">1 ml pipet</td>
			<td style="width:231px;">Fisher</td>
			<td style="width:124px;">13-678-11B</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">5 ml pipet</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:124px;">357543</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">25 ml pipet</td>
			<td style="width:231px;">Ultident</td>
			<td style="width:124px;">DL-357551</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1,5 ml conical polypropylene micro tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">72.690</td>
			<td></td>
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			<td height="21" style="height:21px;width:303px;">15 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.554.205</td>
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			<td height="21" style="height:21px;width:303px;">50 ml conical polypropylene tube</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.547.205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">50 ml high speed polypropylene copolymer tube</td>
			<td style="width:231px;">Nalgene</td>
			<td style="width:124px;">3119-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">250 ml high speed polypropylene bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">356011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay (Bradford assay)</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0006</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protein assay standard II</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0007</td>
			<td></td>
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			<td height="21" style="height:21px;">Test tube 16x100</td>
			<td>VWR</td>
			<td>47729-576</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Test tube 12x75</td>
			<td>Ultident</td>
			<td>170-14100005B</td>
			<td></td>
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			<td height="21" style="height:21px;">Cell incubator&#160;</td>
			<td style="width:231px;">Mandel</td>
			<td style="width:124px;">Heracell 150</td>
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			<td style="width:124px;">Centra8R</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed centrifuge</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Avanti J8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 250ml bottle</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JLA16.250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">High speed rotor for 50ml tube</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JA30.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fow cytometry&#160;</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">FACS Calibur</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Spectrophotometer</td>
			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">Series 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bronchial tissue explants and sections&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice (5-7 weeks old)&#160;&#160;</td>
			<td style="width:231px;">Charles River Laboratories, Inc.&#160;</td>
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			<td></td>
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			<td style="width:124px;">2-82001</td>
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			<td style="width:124px;">371310</td>
			<td>#10</td>
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			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">10003-12</td>
			<td>#7</td>
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			<td style="width:231px;">Olympus Optical CO., LTD.&#160;&#160;&#160;</td>
			<td style="width:124px;">CK2</td>
			<td></td>
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			<td height="25" style="height:25px;">high O2 gas mixture&#160;</td>
			<td>VitalAire Canada Inc.</td>
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			<td height="21" style="height:21px;width:303px;">modular incubator chamber</td>
			<td style="width:231px;">Billups-Rothenberg Inc.</td>
			<td style="width:124px;">MIC-101</td>
			<td></td>
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			<td style="width:231px;">Barnstead Lab-Line,&#160;</td>
			<td style="width:124px;">SHKA4000-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well tissue culture plate</td>
			<td style="width:231px;">Becton Dickinson and Company</td>
			<td style="width:124px;">353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plastic tissue culture dishes (100 mm)</td>
			<td style="width:231px;">Sarstedt, Inc.</td>
			<td style="width:124px;">83.1802</td>
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			<td height="42" style="height:42px;width:303px;">Surgical scissors</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">14060-09</td>
			<td style="width:171px;">Straight, sharp, 9cm longth</td>
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			<td height="21" style="height:21px;width:303px;">Half-curved Graefe forceps</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">11052-10</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:303px;">humidified CO2 incubator</td>
			<td style="width:231px;">Mandel Scientific Company Inc.&#160;</td>
			<td style="width:124px;">SVH-51023421</td>
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			<td style="width:124px;">T555</td>
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			<td style="width:124px;">G6403</td>
			<td></td>
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			<td height="21" style="height:21px;">Cacodylate</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">31533</td>
			<td></td>
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			<td height="21" style="height:21px;">microscope slides</td>
			<td style="width:231px;">VWR Scientific Inc.&#160;</td>
			<td style="width:124px;">48300-025</td>
			<td>25x75mm</td>
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			<td height="21" style="height:21px;">Xylene</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">X5-4</td>
			<td></td>
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			<td height="21" style="height:21px;">Mayer&#39;s hematoxylin</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">MHS16</td>
			<td>Funnel with filter paper&#160;&#160;</td>
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			<td height="21" style="height:21px;">HCl&#160;</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.&#160;&#160;</td>
			<td style="width:124px;">A144s-500</td>
			<td></td>
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			<td height="21" style="height:21px;">eosin&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT110116</td>
			<td>Funnel with filter paper&#160;&#160;</td>
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			<td height="21" style="height:21px;width:303px;">filter paper</td>
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</table>

generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

L&#39;intérêt pour les rôles biologiques des cellules vésicules membranaires dérivés dans la communication cellulaire a augmenté ces dernières années. Des microparticules (MP) sont un tel type de vésicules, de diamètre allant de 0,1 um à 1 um, et typiquement libéré de la membrane plasmique de cellules eucaryotes en cours de l&#39;activation ou de l&#39;apoptose. Nous décrivons ici la génération de microparticules de lymphocytes T dérivés (PMT) à partir de cellules T CEM apoptotiques stimulées par actinomycine D. PMT sont isolés par un processus de centrifugation différentielle en plusieurs étapes et caractérisé par cytométrie de flux. Ce protocole présente également un procédé de détection in situ de la mort cellulaire pour démontrer l&#39;effet pro-apoptotique de PMT sur les cellules épithéliales bronchiques dérivés d&#39;explants de souris primaires de tissus respiratoires bronchiques. Procédés décrits ici prévoient une procédure reproductible pour isoler des quantités abondantes de PMT à partir de lymphocytes apoptotiques in vitro. PMT dérivéde cette manière peut être utilisé pour évaluer les caractéristiques des différents modèles de maladies et de la pharmacologie et de tests de toxicologie. Étant donné que l&#39;épithélium des voies aériennes offre une barrière de protection physique et fonctionnelle entre l&#39;environnement externe et les tissus sous-jacents, l&#39;utilisation d&#39;explants de tissus bronchiques plutôt que des lignées de cellules epitheliales immortalisées fournit un modèle efficace pour les enquêtes qui nécessitent des tissus des voies respiratoires des voies.

PMT ont été caractérisés avec de l&#39;annexine V coloration 10 par la cellule activé par fluorescence (FACS) analyse et fermée en utilisant une um perles dans lequel 97% des députés (≤1 um) ont été annexine-V-Cy5 positive (figure 1A et 1B). Typiquement, environ 2,5 mg de PMT ont été obtenus à la suite de ce protocole. Explants de tissus bronchiques de souris C57BL / 6 ont été soumis à un traitement véhicule et PMT. L&#39;analyse histopathologique des sections bronchiques r...

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

Microparticules membranaires hangar cellulaires (PM) sont des vésicules biologiques actifs qui peuvent être isolés et leurs effets physiopathologiques étudiés dans différents modèles. Nous décrivons ici un procédé pour générer PM dérivées de lymphocytes T (PMT) et pour mettre en évidence leur effet pro-apoptotique sur les cellules epitheliales des voies respiratoires.

Génération de microparticules lymphocytaire et détection de leur effet pro-apoptotique sur les cellules épithéliales des voies aériennes

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

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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-culture des modèles Pseudomonas aeruginosa Les biofilms Cultivé sur le Live cellules des voies respiratoires de l&#39;homme

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

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

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

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

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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

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L&#39;établissement d&#39;une culture liquide recouvert de Airway humaines épithéliales polarisées cellules Calu-3 pour étudier la réponse cellulaire aux agents pathogènes respiratoires hôte<em&gt; In vitro</em

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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

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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

Isolement et caractérisation de microparticules neutrophile dérivés pour des études fonctionnelles

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