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

视力保健研究网络 - 这项工作是由卫生研究所（178918），全宗德RECHERCHE连接桑特魁北克的加拿大学院资助。

注：雄性C57BL / 6小鼠（5-7周龄）从查尔斯河实验室国际公司（ST-恒，魁北克，加拿大），并根据批准的朱圣 - 海宁动物护理委员会的协议操作。小鼠支气管组织的外植体用于研究的LMP的上皮细胞上的促凋亡效应提供初级支气管上皮细胞的良好来源。这个协议描述了在体外产生的LMP的，以及用于检测对LMP的治疗支气管组织的外植体凋亡的上皮细胞的方法。该协议包括3个部分。 <p class="jove_t...

<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>

国会议员是细胞间串扰活性介质和他们的研究是有希望在科学的许多领域。11本研究中提出的体外大规模发电的LMP从凋亡T细胞系衍生的详细协议。这些国会议员表达细胞分子的大型剧目，并且生物牵连的细胞和组织稳态的调节。然而，LMP的源于不同来源可能是生物学上不同。4,9,12,13 的LMP显示根据用于生成它们在体外刺激和从它们所衍生自的细胞?...

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>
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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 style="width:124px;">83.1812.502</td>
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			<td style="width:124px;">13-678-11B</td>
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			<td height="21" style="height:21px;width:303px;">5 ml pipet</td>
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			<td style="width:124px;">357543</td>
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			<td height="21" style="height:21px;width:303px;">25 ml pipet</td>
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			<td style="width:124px;">DL-357551</td>
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			<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 style="width:231px;">Sarstedt</td>
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			<td style="width:231px;">Sarstedt</td>
			<td style="width:124px;">62.547.205</td>
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			<td height="42" style="height:42px;width:303px;">50 ml high speed polypropylene copolymer tube</td>
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			<td height="21" style="height:21px;width:303px;">250 ml high speed polypropylene bottle</td>
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			<td style="width:124px;">356011</td>
			<td></td>
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			<td height="21" style="height:21px;">Protein assay (Bradford assay)</td>
			<td>Bio-Rad Laboratories</td>
			<td>500-0006</td>
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			<td>Bio-Rad Laboratories</td>
			<td>500-0007</td>
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			<td height="21" style="height:21px;">Test tube 16x100</td>
			<td>VWR</td>
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			<td height="21" style="height:21px;">Test tube 12x75</td>
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			<td>170-14100005B</td>
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			<td style="width:124px;">Avanti J8</td>
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			<td style="width:231px;">Beckman</td>
			<td style="width:124px;">JLA16.250</td>
			<td></td>
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		<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>
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			<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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			<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>
			<td style="width:231px;"></td>
			<td style="width:124px;"></td>
			<td></td>
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			<td style="width:231px;">Charles River Laboratories, Inc.&#160;</td>
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			<td></td>
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			<td style="width:231px;">Barnstead Lab-Line,&#160;</td>
			<td style="width:124px;">SHKA4000-7</td>
			<td></td>
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		<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>
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			<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 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;">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;">48300-025</td>
			<td>25x75mm</td>
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			<td style="width:231px;">Fisher scientific&#160; International, Inc.</td>
			<td style="width:124px;">X5-4</td>
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			<td style="width:231px;">Sigma-Aldrich, Inc.&#160;</td>
			<td style="width:124px;">MHS16</td>
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</table>

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

在细胞 - 细胞通讯细胞膜衍生囊泡的生物角色的兴趣，近年来已增加。微粒（MPS）是一种这样的类型的小泡，直径范围为0.1微米至1微米，并且通常从真核细胞进行活化或凋亡的质膜棚。在这里，我们描述了从凋亡的CEM T细胞与放线菌素D的LMP是通过一个多步差速离心处理分离和表征使用流式细胞术刺激的T淋巴细胞衍生的微粒（的LMP）的生成。该协议也为展示的LMP对小鼠原发性呼吸支气管组织植源性支气管上皮细胞凋亡的作用原位细胞死亡检测方法。本文描述的方法在体外分离量的LMP丰富的淋巴细胞凋亡提供了可重复的过程。 LMP的派生以这种方式可以用来评价各种疾病模型的特征，并为药理学和毒理学试验。鉴于气道上皮提供外部环境和下面的组织之间的保护物理和功能障碍，使用支气管组织的外植体，而不是永生化上皮细胞系提供了有效的模型用于要求气道组织的调查。

通过荧光激活细胞分选（FACS）分析，并使用1微米的小珠，其中国会议员（≤1微米）的97％的人膜联蛋白-V-Cy5的阳性门控的LMP进行表征与膜联蛋白V染色10（ 图1A和1B）。通常情况下，的LMP为约2.5毫克，得到下列该协议。从C57BL支气管组织的外植体/ 6小鼠进行车辆和的LMP治疗。支气管切片的组织病理学分析揭示的LMP对支气管上皮细胞的结构完整性的影响。在对照外植体，细支气管...

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

细胞膜棚微粒（MPS）是活性生物囊泡可以分离和它们的病理生理作用研究了各种模型。在这里，我们描述了从生成T淋巴细胞（的LMP）得出国会议员和展示他们对气道上皮细胞凋亡作用的方法。

淋巴细胞微粒和检测其促凋亡作用的气道上皮细胞产生

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

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

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

科研

免疫与感染

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

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

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

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

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

可见光和庆大霉素的协同作用<em&gt;假单胞菌假单胞菌</em&gt;微生物

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

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</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

从人类胸腺髓样树突状细胞和上皮细胞的分离

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

定量检测抗体诱导补体激活的方法对红血细胞

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

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

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

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

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

中性粒细胞衍生微粒的分离与鉴定功能研究

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