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

Este trabalho é apoiado por subsídios da Canadian Institutes of Health Research (178.918), Fonds de recherche en santé du Québec - Visão Rede de Investigação em Saúde.

NOTA: Masculino camundongos C57BL / 6 (5-7 semanas de idade) são de Charles River Laboratories International, Inc. (St-Constant, Quebec, Canadá.) E manipulados de acordo com protocolos aprovados pela Comissão de CHU Sainte-Justine Animal Care. Rato explantes de tecido dos brônquios fornecer uma boa fonte de células epiteliais brônquicas primárias para investigar os efeitos pró-apoptóticos de LMPS em células epiteliais. Este protocolo descreve a geração in vitro de LMPS, bem como um método para dete...

<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>

MPs são mediadores ativos de conversas cruzadas intercelular e seu estudo é promissor em muitas áreas da ciência. 11 Este estudo apresentou um protocolo detalhado para in vitro geração em larga escala de LMPS derivado de uma linha de células T por apoptose. Estas MPs expressar um grande repertório de moléculas de linfócitos e são biologicamente implicado na regulação da homeostase celular e tecidual. No entanto, LMPS derivadas de diferentes fontes podem ser biologicamente diferente. <sup...

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.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">X-VIVO 15 medium&#160;</td>
			<td style="width:231px;">Cambrex, Walkersville</td>
			<td>04-744Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T75</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1813.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Flask T175</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1812.502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Actinomycin&#160;D&#160;</td>
			<td style="width:231px;">Sigma Chemical Co.</td>
			<td style="width:124px;">A9415-2mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PBS</td>
			<td style="width:231px;">Lifetechnologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#181;m filter</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">83.1826.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Annexin-VCy5</td>
			<td style="width:231px;">BD Pharmagen&#160;</td>
			<td>559933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS flow solution</td>
			<td>BD Bio-sciences</td>
			<td style="width:124px;">342003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fluorescent microbeads (1 um)</td>
			<td style="width:231px;">Molecular Probes&#160;</td>
			<td style="width:124px;">T8880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysterene counting beads (7 um)</td>
			<td style="width:231px;">Bangs laboratories</td>
			<td style="width:124px;">PS06N/6994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene FACS tubes</td>
			<td>Falcon</td>
			<td style="width:124px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 16x100</td>
			<td>VWR</td>
			<td>47729-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Test tube 12x75</td>
			<td>Ultident</td>
			<td>170-14100005B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator&#160;</td>
			<td style="width:231px;">Mandel</td>
			<td style="width:124px;">Heracell 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Low speed centrifuge</td>
			<td style="width:231px;">IEC</td>
			<td style="width:124px;">Centra8R</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Airway PrimaCell&#8482; System:</td>
			<td style="width:231px;">CHI Scientific, Inc.</td>
			<td style="width:124px;">2-82001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Rib-Back Carbon Steel Scalpel Blades</td>
			<td style="width:231px;">Becton Dickinson AcuteCare</td>
			<td style="width:124px;">371310</td>
			<td>#10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle</td>
			<td style="width:231px;">Fine Science Tools Inc.&#160;</td>
			<td style="width:124px;">10003-12</td>
			<td>#7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phase-contrast inverted microscope</td>
			<td style="width:231px;">Olympus Optical CO., LTD.&#160;&#160;&#160;</td>
			<td style="width:124px;">CK2</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">high O2 gas mixture&#160;</td>
			<td>VitalAire Canada Inc.</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MaxQ 4000 incubated orbital shaker</td>
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
			<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;">&#160;Histopathological examination&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formalin formaldehyde</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">HT5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">T555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethyl alcohol</td>
			<td style="width:231px;">Merck KGaA, Darmstadt</td>
			<td style="width:124px;">EX0278-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;glutaraldehyde&#160;</td>
			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">G6403</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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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generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

O interesse nas funções biológicas de células derivadas de vesículas de membrana em comunicação célula-célula tem aumentado nos últimos anos. As micropartículas (MP) são um tal tipo de vesículas, que variam em diâmetro de 0,1 mm a 1 mm, e normalmente eliminados da membrana plasmática de células eucarióticas submetidos a activação ou a apoptose. Aqui descreve-se a geração de linfócitos T derivados de micropartículas (LMPS) a partir de células CEM T apoptóticas estimuladas com actinomicina D. LMPS são isoladas através de um processo de múltiplos passos de centrifugação diferencial e caracterizadas por citometria de fluxo. Este protocolo também apresenta um método de detecção da morte celular in situ para demonstrar o efeito pró-apoptótica de LMPS em células epiteliais brônquicas derivadas de rato primários respiratórias explantes de tecido brônquico. Os métodos aqui descritos proporcionam um procedimento reprodutível para o isolamento de quantidades abundantes de LMPS de linfócitos apoptóticos in vitro. LMPS derivadodeste modo pode ser utilizado para avaliar as características de vários modelos de doenças, e para a farmacologia e toxicologia de teste. Dado que o epitélio das vias aéreas como uma barreira física e funcional protectora entre o ambiente externo e o tecido subjacente, o uso de explantes de tecido brônquico, em vez de linhas de células epiteliais imortalizadas fornece um modelo eficaz para investigações que requerem tecido vias aéreas.

LMPS foram caracterizados com coloração de anexina V por 10 células activadas por fluorescência (FACS) e fechado utilizando uma pm grânulos em que 97% das MPs (≤1 um) foram anexina-V-Cy5 positivo (Figura 1A e 1B). Normalmente, cerca de 2,5 mg de LMPS foram obtidos seguindo este protocolo. Explantes de tecido brônquico de ratos C57BL / 6 foram submetidos a tratamento e LMPS veículo. A análise histopatológica das secções brônquicas revelado o efeito de LMPS sobre a integridade est...

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

Micropartículas verter à membrana celular (MPs) são vesículas biológicas activas que podem ser isolados e os seus efeitos patofisiológicos investigados em vários modelos. Descrevemos aqui um método para a geração de MPs derivadas de linfócitos T (LMPS) e para demonstrar o seu efeito pró-apoptóticos em células epiteliais das vias respiratórias.

Geração de Lymphocytic micropartículas e Detecção de seu efeito pró-apoptóticos em células epiteliais das vias aéreas

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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