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。 1.生产的LMP与表征注意:为防止污染，确保在这个实验中使用的所有材料是无菌的或高压灭菌。执行所有的步骤，在RT在生物安全柜无菌条件下，除非另有说明。 1.1）刺激和收集的国会议员9 <ol><li>解冻千万CEM T细胞等分在37℃水浴。稀释在10ml预热的无血清造血介质如X-VIVO，在15ml无菌管中并离心，在200 GX 5分钟。吸在5ml预热的培养基上清液和重悬细胞。 </li><li>转移细胞到T75组织培养瓶（对于悬浮细胞）用15ml预热的造血介质如X-VIVO并在湿润的培养箱中孵育4天，在37℃，5％的CO 2。 </li><li> 4天后，转移所有的培养基和细胞分化成含有100ml新鲜培养基一个T175组织培养瓶。继续孵育细胞在相同的条件下约72小时，直到它们长到200万个细胞/ ml的密度。 </li><li>均匀4 T175瓶中各含有150ml新鲜培养基，并继续培养细胞直到细胞已经生长（约48小时温育），以200万/ ml的密度之间分裂的细胞。 </li><li&gt;通过离心，在200×g离心收集从每个烧瓶中的细胞5分钟，并悬浮300×10 6细胞到含有150ml新鲜培养基的新T175烧瓶中，以维持2000000 / ml的细胞密度。 </li><li>加入放线菌素D（溶解在DMSO中，在2毫克/毫升）的培养基中以0.5微克/ ml的终浓度，并培育24小时。 </li><li>传送所有的培养基到50ml锥形管中，并降速细胞在750×g离心5分钟。转移上清液入50ml锥形管中并离心，在1500×g离心15分钟以除去大细胞碎片。 </li><li>转移上清液到250ml瓶中，超速离心机在12,000×g离心50分钟。弃去上清液，并收集颗粒。 </li><li>洗的LMP富集粒料用40ml无菌PBS在50ml管离心12000×g离心50分钟。重复此步骤两次。 </li><li>收集最后一次洗涤上清液;它将被用来作为载体对照。暂停的LMP颗粒1毫升的PBS并转移到1.5毫升无菌微型管。分装和储存孤立的LMP在-80°C（避免多重自由解冻周期）。 </li></ol>国会议员1.2），通过流式细胞仪分析4表征<ol><li>制备2个样品的膜联蛋白缓冲液中，用1和另一无CaCl 2:的Hepes的10mM，NaCl的140mM的，加或减5毫米氯化钙2。 </li><li>用0.22微米的过滤器以除去颗粒过滤膜联蛋白缓冲液和FACS流动鞘液。 </li><li>稀释1微升的LMP在44微升膜联蛋白缓冲液用5mM的CaCl 2成在FACS管中。准备另一管1微升的LMP中44微升膜联蛋白缓冲液无CaCl 2（阴性对照）。 </li><li>添加5微升annexinV-Cy5的在每个管拌匀。孵育在室温下15分钟，在黑暗中。停止反应稀释用400μl的FACS流动鞘液在每个管中混合。 </li><li>加入的7微米计BEA 10微升（200,000珠）DS悬浮在每个管内标，以获得绝对计数。 </li><li>建立相对大小门（FSC-H，PMT E00，对数刻度）和相对粒度（SSC-H，PMT 325，对数刻度）点图上用流式细胞仪的尺寸校准荧光珠流1微米（1号门）和算珠门7微米（2号门）。 </li><li>分析利用已建立的门和FL-4通道的膜联蛋白FSC-H / SSC-H情节的LMP样品（PMT 765，对数刻度）点图，通过收购一个信号，直到20000计数珠在2号门的到达。 </li><li>确定在含有氯化钙的膜联蛋白缓冲液的LMP的正annexinV事件，然后减去的LMP中的膜联蛋白缓冲液中的事件无CaCl 2（阴性对照）。 </li><li>计算根据下列方程式国会议员的绝对数量: <img alt="式（1）" fo:content-width="4in" src="/files/ftp_upload/52651/52651eq1.jpg" /></li></ol> 1.3）测定MP P的rotein浓度（Bradford法） <ol><li>制备从1.25至20微克/毫升的蛋白质标准的5系列稀释。吸移管800微升的每个标准和样品溶液注入一式两份一个干净的试管中。加入200μl布拉德福德染料试剂到每个管中。拌匀，然后在室温下孵育5分钟。 </li><li>测定吸光度在595nm处。确定的LMP使用标准曲线的线性回归中的蛋白质浓度。 </li></ol> 2.支气管组织植和治疗的LMP 注:要特别注意无菌的工作​​环境，无菌准备在下面的实验中使用的解决方案和媒介。准备完全愈合中，加入1毫升组织愈合中补充与血清（解冻冰）至100毫升组织愈合中拌匀。 2.1）准备支气管组织植<ol><li>培养之前，划伤1厘米2 6区每个在每100mm组织培养皿用手术刀叶片的表面的边缘。涂层的每个刮擦100mm组织培养皿用2ml培养皿涂布溶液，并孵化培养皿中的潮湿的 CO 2培养箱中O / N在37℃。真空吸出多余的解决方案，填补了菜用15毫升组织清洗介质。 </li><li>根据批准的动物护理伦理委员会的协议由CO安乐死C57BL / 6小鼠（5〜7周龄）2吸入。 </li><li>无菌解剖肺组织手术刀，杜蒙超细镊子和手术剪。小心地取出实质和血管。放置肺组织入冰冷的组织洗涤培养基运输到实验室，如果适用的话。 </li><li>进一步解剖支气管浸没在组织培养基洗涤并分离支气管直径为1到2.5mm，从外周肺组织。切片支气管组织成约5毫米厚的支气管环用手术刀。 </li&gt; <li>使用的无菌弯microdissecting镊子掏挖的议案，拿起支气管片段并将其放置到的菜划伤的地方。 </li><li>取出组织洗涤培养基，孵育所述片段在RT〜5分钟，以使它们附着在菜肴。 </li><li>加入10 mL完全愈合中的每道菜，并放置在一个可控气氛模块化孵化室。冲洗具有高的 O 2的混合气体（70％O 2，25％N 2和5％的CO 2，）的腔室。放置室在台式轨道培养箱，在37℃下摇晃。摇动室放置24小时以每分钟10个循环，以允许介质流间歇以上的片段。 </li><li>经过24小时培养后，观察在相差倒置显微镜下的组织外植体。选择支气管外植体完整，汗毛运动与活泼的支气管上皮细胞进行后续的LMP治疗。 </li></ol>2.2）的LMP治疗<ol><li>制备完全生长培养基如下:解冻生长培养基补充剂在冰上血清和成纤维细胞抑制剂。加入1毫升的生长培养基补充有血清和200μl的成纤维细胞抑制剂至100ml生长培养基;调匀。升温，在37℃的完全生长培养基在使用前10分钟。 </li><li>稀分离的LMP在一个新的无菌微量离心管中，用PBS以制备的LMP股票以800微克/毫升的浓度。 </li><li>加入0.5毫升完全生长培养基中以12孔组织培养板的各孔中。 </li><li>传送与从先前的协议（第2.1节），以在组织培养板的各孔的弯曲microdissecting钳子所选支气管植。 </li><li>适当标记培养板，以确定的LMP处理井和控制井。添加25微升的LMP库存入每个的LMP处理井（对于40微克/ ml的终浓度）和25μl的对照载体（见LMP<html>的生产），以对照孔。 </li><li>继续温育在受控气氛模块化培养箱室中于37℃轻轻摇动。 </li><li> 24小时后，洗涤外植体3次，用PBS中并继续进行下一步骤（4％多聚甲醛[PFA]的固定）。 </li></ol> 3.组织病理学检查 3.1）准备了以下解决方案之前，进行下一个步骤<ol><li>通过混合137 mM氯化钠，2.7 mM的氯化钾，10毫的Na 2 HPO 4，1.76毫KH 2 PO 4，pH 7.4中制备的1×PBS缓冲液中。 </li><li>以制备4％PFA，溶解在400ml水中，在60℃下加热搅拌20克煤灰;加入几滴10 M氢氧化钠以清除溶液。接下来添加1X PBS缓冲和调节音量到500毫升pH值至7.4。筛选和分装;储存在-20℃。 </li><li>准备以下脱水或补液试剂; 100％，90％，70％，50％的乙醇和二甲苯。 </li></ol>e_step"&gt; 3.2）外植体固定，并组织部分脱蜡<ol><li>放置在一个标记的离心管中每一个外植体用1.5ml 4％的PFA和孵化O / N在4℃。与1X PBS冲洗两次植。 </li><li>通过醇系列脱水外植体（70％乙醇:每3次30分钟; 90％乙醇:每2次30分钟; 100％乙醇:3次每次30分钟;然后二甲苯:3次，每次20分钟，每一个）。执行在通风橱中的所有步骤，在RT。 </li><li>在烘箱中嵌入组织外植体中的石蜡在58℃。用旋转切片机准备5微米厚的组织切片。 </li><li>漂浮在56℃水浴的部分，然后装入部分标上组织幻灯片。放置在手动染色机架和干燥载玻片在65℃下1小时。允许滑动以冷却在RT。 </li><li>浸在含有二甲苯，每次10分钟，以去除石蜡连续4次污点菜架。浸齿条在乙醇系列以去除二甲苯:100％，然后95％，第烯80％，然后70％，然后50％乙醇（5分钟，每步）。用自来水冲洗齿条5分钟，以除去乙醇。 </li></ol> 3.3），HE染色（H＆E）染色<ol><li>继续与固定的组织切片工作;齿条放入一个染色皿填充用Mayer氏苏木素15分钟。冲洗齿条用自来水以除去苏木20分钟。 </li><li>放置在蒸馏水中进行30 sec.Place在95％乙醇中30秒。放置在曙红Y溶液染色皿1分钟。脱水，通过对每个2分钟2的变化的95％乙醇，100％乙醇和二甲苯。 </li><li>在显微镜下进行快速检查，以确保剩余的曙红被除去。放置2〜3滴封固剂（费希尔SP15-100）的到每个滑动，然后盖上盖玻璃。 </li></ol> 3.4） 原位细胞凋亡检测:TUNEL法<ol><li>在开始之前，请准备蛋白酶K工作液:20微克/毫升10毫米的Tris /盐酸，pH值7.4。 </li><li>重复步骤1至5节3.2（植固定，组织切片脱蜡）的。冲洗用去离子H 2 O的幻灯片</li><li>浸入用1×PBS的幻灯片10分钟。沥干多余的PBS。孵育组织切片30分钟，在RT下用蛋白酶K工作溶液。冲洗1X PBS滑两次。 </li><li>细胞死亡检测试剂盒的使用说明书中所述进行TUNEL法。安装使用安装介质中，并与盖玻片盖玻片手动。 </li><li>在光学显微镜下分析样品。使用图像Pro的4.5来分析细胞凋亡褐色。 </li></ol>

<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. 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作者什么都没有透露。

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.

<table><tbody><tr><td>LMPs production and characterization</td><td></td><td></td><td></td></tr><tr><td>CEM T cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CCL-119</td><td></td></tr><tr><td>X-VIVO 15 medium&amp;nbsp;</td><td>Cambrex, Walkersville</td><td>04-744Q</td><td></td></tr><tr><td>Flask T75</td><td>Sarstedt</td><td>83.1813.502</td><td></td></tr><tr><td>Flask T175</td><td>Sarstedt</td><td>83.1812.502</td><td></td></tr><tr><td>Actinomycin&amp;nbsp;D&amp;nbsp;</td><td>Sigma Chemical Co.</td><td>A9415-2mg</td><td></td></tr><tr><td>PBS</td><td>Lifetechnologies</td><td>14190-144</td><td></td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>Annexin-VCy5</td><td>BD Pharmagen&amp;nbsp;</td><td>559933</td><td></td></tr><tr><td>FACS flow solution</td><td>BD Bio-sciences</td><td>342003</td><td></td></tr><tr><td>Fluorescent microbeads (1 &amp;micro;m)</td><td>Molecular Probes&amp;nbsp;</td><td>T8880</td><td></td></tr><tr><td>Polysterene counting beads (7 &amp;micro;m)</td><td>Bangs laboratories</td><td>PS06N/6994</td><td></td></tr><tr><td>Polypropylene FACS tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>1 ml pipet</td><td>Fisher</td><td>13-678-11B</td><td></td></tr><tr><td>5 ml pipet</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>25 ml pipet</td><td>Ultident</td><td>DL-357551</td><td></td></tr><tr><td>1.5 ml conical polypropylene micro tube</td><td>Sarstedt</td><td>72.690</td><td></td></tr><tr><td>15 ml conical polypropylene tube</td><td>Sarstedt</td><td>62.554.205</td><td></td></tr><tr><td>50 ml conical polypropylene tube</td><td>Sarstedt</td><td>62.547.205</td><td></td></tr><tr><td>50 ml high speed polypropylene copolymer tube</td><td>Nalgene</td><td>3119-0050</td><td></td></tr><tr><td>250 ml high speed polypropylene bottle</td><td>Beckman</td><td>356011</td><td></td></tr><tr><td>Protein assay (Bradford assay)</td><td>Bio-Rad Laboratories</td><td>500-0006</td><td></td></tr><tr><td>Protein assay standard II</td><td>Bio-Rad Laboratories</td><td>500-0007</td><td></td></tr><tr><td>Test tube 16 x 100</td><td>VWR</td><td>47729-576</td><td></td></tr><tr><td>Test tube 12 x 75</td><td>Ultident</td><td>170-14100005B</td><td></td></tr><tr><td>Cell incubator&amp;nbsp;</td><td>Mandel</td><td>Heracell 150</td><td></td></tr><tr><td>Low speed centrifuge</td><td>IEC</td><td>Centra8R</td><td></td></tr><tr><td>High speed centrifuge</td><td>Beckman</td><td>Avanti J8</td><td></td></tr><tr><td>High speed rotor for 250ml bottle</td><td>Beckman</td><td>JLA16.250</td><td></td></tr><tr><td>High speed rotor for 50ml tube</td><td>Beckman</td><td>JA30.50</td><td></td></tr><tr><td>Fow cytometry&amp;nbsp;</td><td>BD Bio-sciences</td><td>FACS Calibur</td><td></td></tr><tr><td>Spectrophotometer</td><td>Beckman</td><td>Series 600</td><td></td></tr><tr><td>Bronchial tissue explants and sections&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6 mice (5-7 weeks old)&amp;nbsp;&amp;nbsp;</td><td>Charles River Laboratories, Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Mouse Airway PrimaCell&amp;trade; System:</td><td>CHI Scientific, Inc.</td><td>2-82001</td><td></td></tr><tr><td>&amp;nbsp;Rib-Back Carbon Steel Scalpel Blades</td><td>Becton Dickinson AcuteCare</td><td>371310</td><td>#10</td></tr><tr><td>Scalpel Handle</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>10003-12</td><td>#7</td></tr><tr><td>phase-contrast inverted microscope</td><td>Olympus Optical CO., LTD.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>CK2</td><td></td></tr><tr><td>high O&lt;sub&gt;2&lt;/sub&gt; gas mixture&amp;nbsp;</td><td>VitalAire Canada Inc.</td><td></td><td></td></tr><tr><td>modular incubator chamber</td><td>Billups-Rothenberg Inc.</td><td>MIC-101</td><td></td></tr><tr><td>MaxQ 4000 incubated orbital shaker</td><td>Barnstead Lab-Line,&amp;nbsp;</td><td>SHKA4000-7</td><td></td></tr><tr><td>12-well tissue culture plate</td><td>Becton Dickinson and Company</td><td>353043</td><td></td></tr><tr><td>Plastic tissue culture dishes (100 mm)</td><td>Sarstedt, Inc.</td><td>83.1802</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>14060-09</td><td>Straight, sharp, 9cm longth</td></tr><tr><td>Half-curved Graefe forceps</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>11052-10</td><td></td></tr><tr><td>humidified CO2 incubator</td><td>Mandel Scientific Company Inc.&amp;nbsp;</td><td>SVH-51023421</td><td></td></tr><tr><td>&amp;nbsp;Histopathological examination&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>formalin formaldehyde</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>HT5011</td><td></td></tr><tr><td>paraffin</td><td>Fisher scientific&amp;nbsp; International, Inc.</td><td>T555</td><td></td></tr><tr><td>ethyl alcohol</td><td>Merck KGaA, Darmstadt</td><td>EX0278-1</td><td></td></tr><tr><td>&amp;nbsp;glutaraldehyde&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>G6403</td><td></td></tr><tr><td>Cacodylate</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>31533</td><td></td></tr><tr><td>microscope slides</td><td>VWR Scientific Inc.&amp;nbsp;</td><td>48300-025</td><td>25x75 mm</td></tr><tr><td>Xylene</td><td>Fisher scientific&amp;nbsp; International, Inc.</td><td>X5-4</td><td></td></tr><tr><td>Mayer&#039;s hematoxylin</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>MHS16</td><td>Funnel with filter paper&amp;nbsp;&amp;nbsp;</td></tr><tr><td>HCl&amp;nbsp;</td><td>Fisher scientific&amp;nbsp; International, Inc.&amp;nbsp;&amp;nbsp;</td><td>A144s-500</td><td></td></tr><tr><td>eosin&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>HT110116</td><td>Funnel with filter paper&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Permount&amp;trade; Mounting Medium</td><td>Thermo Fisher Scientific Inc.&amp;nbsp;</td><td>SP15-100</td><td></td></tr><tr><td>glass coverslip</td><td>surgipath medical industries, Inc.</td><td>84503</td><td>24&amp;times;24&amp;nbsp;#1&amp;nbsp;</td></tr><tr><td>TUNEL detection kit</td><td>In Situ Cell Death Detection, POD</td><td>11 684 817 910</td><td></td></tr><tr><td>oven</td><td>Despatch Industries Inc.</td><td>LEB-1-20</td><td></td></tr><tr><td>rotary Microtome</td><td>Leica Microsystems Inc.</td><td>RM2145</td><td></td></tr><tr><td>filter paper</td><td>Whatman International Ltd.</td><td>1003150</td><td>#3</td></tr><tr><td>Microscope</td><td>Nikon Imaging Japan Inc.</td><td>E800</td><td></td></tr><tr><td>staining dish complete</td><td>Wheaton Industries, Inc.</td><td>900200</td><td>including dish, rack, cover</td></tr><tr><td>1.5 ml eppendorf tube</td><td>Sarstedt Inc.&amp;nbsp;</td><td>72.69</td><td>39x10 mm</td></tr><tr><td>Orbital and Reciprocating Water Bath</td><td>ExpotechUSA</td><td>ORS200</td><td></td></tr><tr><td>phosphate buffered saline&amp;nbsp;&amp;nbsp;</td><td>GIBCO</td><td>14190-144</td><td></td></tr><tr><td>fume hood</td><td>Nicram RD Service</td><td>3707E</td><td></td></tr></tbody></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 ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.9K 次观看.  University of Montréal. 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。 

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

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

科研

免疫与感染

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

生成人诱导的多能干细胞衍生的2型肺泡上皮细胞的3D球体和2D气液界面培养物

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

发育生物学

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

从人多能干细胞中产生气道上皮细胞气液界面培养物

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

生物学

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...