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/pubmed?term=((Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease%3A+friend+or+foe%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">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/pubmed?term=((Microparticles%3A+targets+and+tools+in+cardiovascular+disease%5BTitle%5D)+AND+%22Trends+Pharmacol+Sci%22%5BJournal%5D)">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/pubmed?term=((Therapeutic+potential+of+plasma+membrane-derived+microparticles%5BTitle%5D)+AND+%22Pharmacol+Rep%22%5BJournal%5D)">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/pubmed?term=((Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">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/pubmed?term=((Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells%5BTitle%5D)+AND+%22Cancer+Biol+Ther%22%5BJournal%5D)">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/pubmed?term=((Leukocyte-derived+microparticles+in+vascular+homeostasis%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">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/pubmed?term=((CD8%2B+T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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>
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</ol>

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

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

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

科研

生物工程

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

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

免疫与感染

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 ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

在合并臭氧和LPS诱导穆林急性肺损伤期间可视化肺细胞适应

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

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

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

医学

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 ...

分析 THP-1 细胞中 LPS/ATP 触发的细胞死亡机制

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show&#160;that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

作者聚焦:THP-1 巨噬细胞对 LPS/ATP 的反应 — 揭示细胞焦亡、细胞凋亡和坏死性凋亡谱

This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

LPS和ATP诱导的PMA分化THP-1巨噬细胞死亡及其验证

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced ...

淋巴细胞微粒和检测其促凋亡作用的气道上皮细胞产生

摘要

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LPS和ATP诱导的PMA分化THP-1巨噬细胞死亡及其验证