Name: investigating mast cell secretory granules; from biosynthesis to exocytosis
Uploaded: 2015-01-26T14:00:00.000+00:00
Duration: 16 min 1 s
Description: Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

我们感谢U.阿瑟瑞博士NPY-MRFP的cDNA的礼物。我们感谢博士。 MJ Kofron，L. Mittleman，M. Shaharbani和Y. Zilberstein与显微镜宝贵援助和图像分析。我们也感谢约瑟夫·奥利博士这个手稿的批判性阅读。这项工作是由以色列科学基金会的资助，由以色列科学院科学支持成立（1139至1112年，以RS-E）。

1.准备RBL细胞培养基<ol><li>混合500毫升低葡萄糖Dulbecco改进的Eagle培养基（DMEM）与56毫升胎牛血清（这使得10％FBS），然后添加5.5青霉素链霉素毫升（这使得〜1％PEST）。 </li><li>用500-1000毫升瓶顶真空过滤器具有0.22微米的孔径大小和存储在4℃过滤介质。 </li></ol> 2.文化RBL细胞<ol><li>在任一板或培养瓶生长RBL细胞在5％的CO 2的潮湿气氛中在37℃。 RBL细胞正在生长于10cm板应补充有10毫升培养基，形成的〜10 7个细胞/板汇合单层。 </li><li>当细胞层将至合流，replate培养以较低的密度。 <ol><li>除去从板/烧瓶用无菌巴斯德移液管连接到真空中的培养基中。 </li><li>冲洗细胞单层用预热（37℃）0.25％胰蛋白酶/ EDTA，涵盖了整个单层（体积取决于培养板/瓶的大小;2毫升为10cm平板）。这将分离的细胞。 </li><li>或者，冲洗细胞单层用磷酸盐缓冲盐水（PBS），除去PBS并加入预热（37℃）胰蛋白酶/ EDTA。这一步分离的细胞快。 </li><li>放置板/烧瓶在恒温箱在37℃（不超过10分钟）。 </li><li>检查该光学显微镜下的细胞，以检查它们是否已​​经脱离。如果细胞10分钟后没有脱落，轻轻拍打上盘/瓶。 </li><li>添加培养基向培养板/烧瓶以悬浮细胞（该介质的体积应至少2倍比胰蛋白酶溶液的体积大）。 </li><li>沉淀细胞离心3分钟，以200g在25℃。 </li><li>重悬在培养基中的细胞。由1:10稀释的细胞和种子在一个新的培养皿中。孵育细胞48-72小时，直到他们REACħ大约90％汇合。 </li><li>长达3个月（30代）重复相同的过程（部分2.2.1-2.2.8）。 </li></ol></li></ol> 3.准备转染媒体。 <ol><li>制备K -管道pH值7， 钙离子乙酸1mM溶液和镁离子醋酸100mM的溶液的100毫溶液。 </li><li>稀释的K-管道的pH 7的100mM的溶液成使用DMEM培养基20毫米， 钙醋酸1mM溶液添加至10微米，镁离子醋酸100mM的溶液的最终浓度为2的终浓度毫米。 </li><li>权衡谷氨酸钾，并添加到该溶液中，以128毫米的最终浓度，拌匀，并保持在4℃直至使用。保持其余的解决方案（ 即 ，K -管道， 钙离子，乙酸镁离子乙酸酯）于-20℃。 </li></ol> 4.转染RBL细胞<ol><li>从使用该板/烧瓶除去培养基无菌巴斯德吸管连接至真空。 </li><li>冲洗细胞单层用预热（37℃）胰蛋白酶/ EDTA应覆盖整个单层。放置板/烧瓶在恒温箱在37℃（不超过10分钟）。检查，如果细胞用光学显微镜已经分离。 </li><li>添加培养基向培养板/烧瓶以悬浮细胞（在介质的体积应至少2大于胰蛋白酶溶液的体积更大）。 </li><li>使用血细胞计数器计数细胞数。 </li><li>沉淀1.5×10 7个细胞离心3分钟，在200×g离心25℃。弃去上清液，加入280微升转介质。转移反应混合物成4mm杯。 </li><li>加入20 NPY-MRFP质粒微克，要么30微克控制空质粒或测试质粒。该反应混合物的最终体积应为300微升。 </li><li>立即放置在冰上10分钟。擦去残留的水和对反应杯roceed电穿孔在300V 9毫秒。 </li><li>为胞吐的测量，在含有300微升培养基的24孔组织培养皿中立即replate细胞。加入8微升的反应混合物（4×10 5细胞）至各孔。加入4×10 5个非转染细胞额外井控制。准备足够的孔中至少有两个孔，每个处理为每转染。 </li><li>对于时间推移显微镜，在含有80微升培养基的8孔腔室硼硅酸盐玻璃罩系统立即replate细胞。添加1.5微升的反应混合物（7.5×10 4个细胞）至各室。 （尽量避免使用该室在玻璃罩的边缘，该室在玻璃罩的中心更容易图像）。 注:重要的是，并非所有的细胞响应于触发和偶尔的时间推移显微镜是由于失去焦点的8孔腔室borosi和漂移废止licate玻璃罩系统。因此，准备至少8室，每个处理每个转染。 </li><li>对于敏化的细胞为了FcεRI介导的活化，加入小鼠DNP特异性单克隆IgE的1微克/毫升到介质（孵育的细胞的IgE至少2小时）。 </li><li>后18-24小时用荧光显微镜，以确认该细胞表达的质粒。 MRFP荧光的激发和发射波长分别是:λEx584，λEm607纳米。 NPY-MRFP应该出现在对应于SGS的囊泡结构。如果感兴趣的第二个基因融合到荧光标签使用荧光显微镜来确认的共表达两种质粒在相同的细胞。例如，如果所测试的基因融合到绿色荧光蛋白，GFP荧光的激发和发射波长分别是:λEx488，λEm507纳米。绿色荧光蛋白标记的蛋白质应出现在表达NPY-MRFP相同的细胞。 </li><li>对于胞外分泌的测量进行吨Ø第5步和时间的推移显微镜，继续执行步骤6。 </li></ol> 5.测量NPY-MRFP胞吐作用<ol><li>制备台氏缓冲液: <ol><li>制备的54毫米氯化钾，20mM的MgCl 2的，2.74 M氯化钠和8毫的NaH 2 PO 4在DDW中的溶液。拌匀，保存在4℃。本步骤是制备的20倍的Tyrode缓冲液中。 </li><li>制备的20毫摩尔的Hepes中的溶液pH值为7，1.8毫氯化钙 ，1毫克/毫升BSA，5.6毫葡萄糖和1至20的稀释20倍的Tyrode在DDW中并充分混合。本步骤是制备1X的Tyrode缓冲液中。 </li><li>分装1X的Tyrode缓冲液并储存在-20℃。避免反复冻融。 </li></ol></li><li>除去从24孔板的培养基中洗3次的Tyrode缓冲液中。通过加入200微升的Tyrode缓冲液以控制井制备未刺激细胞。 </li><li>制备10微升/孔20X的浓缩活化试剂〔（ 例如 1000纳克/毫升的DNP-BSA或DNP-HSA（银），200μM的Ca 2+离子载体（例如 A23187），和20微米的Ca 2+离子载体和1000纳米12-O-十四烷-13-乙酸酯（TPA）]的组合。如果试剂被储存在DMSO中，稀释试剂成20倍的浓度中含有1％DMSO中的Tyrode缓冲液中。孵育在37℃下30分钟。 </li><li>以及小心取出每个上清液至96孔板，放置在冰上，并避免光照。 （上清液包含所述嵌合肽的NPY-MRFP被从细胞中释放）。 </li><li>加含有0.5％的Triton X-100，以每200微升的Tyrode缓冲液，并培育在37℃下进行10分钟。 （本步骤是制备包含NPY-MRFP为没有从细胞中释放的剩余的细胞裂解物的重要）。收集细胞裂解物，并转移到一个96孔板中，放置在冰上，并避免光照。 </li><li>使用fluoresc测量细胞上清液和细胞裂解物的荧光ENCE板读数器，使用的是590-，20纳米的带宽激发滤光片和635-，35纳米的带宽发射滤光片。 </li><li>计算NPY-MRFP公布的百分比: 注:荧光读取器测量任意荧光单位（AFU）。 AFU值取决于机器和它的灵敏度和转染效率上。 <ol><li>设置未转染的RBL细胞作为空白的自体荧光。将每个上清液的总荧光的AFU（上清液+ AFU相应裂解物的AFU）再乘以100。 </li></ol></li></ol>胞吐6.定时显微镜<ol><li>在8孔室硼硅酸盐玻璃罩系统种子7.5×10 4个转染细胞/室。后18-24小时，从所述腔室取出培养基洗3次，用蒂罗德缓冲</li><li>加入72微升的Tyrode缓冲液到每个室中。淡化台氏缓冲的激活剂，以10倍的浓度。 </li> &lt;li&gt;使用配备有加热室（37°C）和CO 2控制器（4.8％）和40X或63X客观的共聚焦荧光显微镜。打开显微镜系统:汞灯，计算机，激光器。确保该加热室是在合适的温度在开始实验之前。 </li><li>将室中的加热室中，并确保该室被正确和稳定的安装。 </li><li>根据相关的荧光团开启荧光灯和可视化转染的细胞。关闭荧光一旦感兴趣的细胞是在现场，以尽量减少漂白和细胞毒性。这个字段将包含约2-3转染的细胞是重要的。转染的细胞应良好分散，但不互相接触。 </li><li>调整激光功率（取决于显微镜）以减少噪声和过饱和，以及毒性，并设置增益和偏移修改信噪比。在扫描速度快或德，以尽量减少激光博览会（平均2）的持续时间。 </li><li>调节针孔尺寸到最大，这使得能够降低激光功率，并保持集中的时间很长的周期的图像。如果需要的话，设置参数为Z-堆栈来重建图像在三维，调整针孔大小为1通风单元（AU），该给出最佳的信噪比，并获得连续的二维共焦光学切片扫描ž-axes光学切片≤0.7微米。 </li><li>设定每个采集到15-30秒，总采集到15分钟的持续时间之间的间隔时间，并开始获取图像。 5分钟后，采集的暂停时间序列。添加8微升10倍的触发，并立即继续进行收购。 </li><li>保存的图片，采用了卷积软件进行反褶积和重​​建书库三维图像，并使用了Imaris软件的一部电影。 </li></ol><ol><li>从共聚焦荧光显微镜来了Imaris软件获得的解卷积时间序列图像导入数据。该软件可读取超过40显微镜文件。如果软件无法读取的文件;将文件转换为TIF文件和进口。 </li><li>时间点添加到一个开放的数据集新闻编辑结束 - &gt;添加时间点，并导入新的数据集。 </li><li>创建使用表面向导选项MRFP通道的表面。选择默认的算法。选择的NPY-MRFP的信道。纪念平滑选项，以减少噪音。为了避免损失的小细节降低区域的细节层面，以0.05微米。 </li><li>设定强度阈值。新的灰面将显示。进入"设置"，并从"中心点"到"面"切换风格。 </li><li>转到所选对象的属性的统计选项卡中，选择详细的标签和特定值SUCH作为荧光强度，音量等。 </li><li>检查数据并确认值与图像相兼容。例如，两个或更多个相邻的颗粒可能被测量为1大颗粒。用于量化的平均颗粒尺寸除以合并的颗粒，以颗粒剂的实际数量的大小。 </li><li>导出所需的数据集到Excel文件和分析数据。 </li></ol> ˚F

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P., Matsui, T., Fukuda, M., Sagi-Eisenberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+regulation+of+mast+cell+exocytosis+by+networks+of+Rab+GTPases.">Decoding the regulation of mast cell exocytosis by networks of Rab GTPases.</a> J Immunol. 189 (5), 2169-2180 (2012).</li><li>Stenmark, H., 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=Inhibition+of+rab5+GTPase+activity+stimulates+membrane+fusion+in+endocytosis.">Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.</a> EMBO J. 13 (6), 1287-1296 (1994).</li><li>Azouz, N. P., 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=Rab5+is+a+novel+regulator+of+mast+cell+secretory+granules:+impact+on+size,+cargo,+and+exocytosis.">Rab5 is a novel regulator of mast cell secretory granules: impact on size, cargo, and exocytosis.</a> J Immunol. 192 (9), 4043-4053 (2014).</li></ol>

The authors have no financial conflicts of interest.

我们描述了一种创新的策略，通过时间流逝立体使用报道基因用于胞吐作用在活细胞中的SG成像结合的MC胞吐和4（X，Y，Z，t）的维数的量化值的量化。这项技术使蛋白质他们对MC功能影响的家庭，如监测的SG，早在他们从高尔基通过其成熟的退出，收购胞外分泌能力和脱粒开始筛选。胞吐连同了SGS的形态和动能表征测量相结合，提供了深入了解通过该测试的蛋白介导MC功能的机制。因此，使用这种?...

肥大细胞（MC）是最知名的是它们在过敏性和炎性反应，如关节炎，哮喘，嗜酸细胞性食管炎，慢性皮炎和过敏性休克1,2-以及其他疾病，包括冠状动脉疾病3,5-和参与免疫细胞癌症3,4。此外，管委会发挥固有免疫和适应性免疫的重要角色，无论是在对抗细菌和寄生虫的宿主防御和抑制免疫反应，例如诱导移植耐受5,6。 的MC从骨髓来源，来自CD34 + / CD117 +多潜能祖细胞7显影。致力于骨髓祖细胞的MC被释放到血液中，并迁移到结缔组织和上皮表面8内定位主要的外周组织。成熟和终末分化的影响o在最终会实现f显示周围环境8,9内细胞因子。 管委会可通过过敏原（抗原，抗原），其遭遇导致免疫球蛋白E（IgE）的一代型抗体被激活。这样的IgE结合到MC的了FcεRI受体，随后交联的细胞结合的IgE的经重新暴露于相同的银，导致了FcεRI聚集和一个信号级联高潮在细胞脱粒的起始[10,11中综述] 。的MC也被激活，独立的IgE，通过神经肽5,12，毒素13，细菌和病毒抗原14,15，多个带正电荷的肽统称为基本分泌素，免疫细胞和细胞因子5,13,12,16的17。的MC是由许多自身释放介质，其进一步放大炎性反应也被激活。 管委会都挤满了分泌颗粒（SGS）含有免疫调节介质，包括血管活性胺，如组胺和血清素（在啮齿动物），蛋白聚糖，蛋白酶，如胃促胰酶和类胰蛋白酶，血管内皮生长因子和多种细胞因子和趋化因子8,9。这些介质是"准备好"，并一次的MC是由一个适当的刺激物激活，这些介质被从细胞通过在几秒钟到几分钟18,19事项调节胞吐作用（脱粒）释放。这个初始事件之后是从头合成一个大阵列的生物效物质，包括花生四烯酸代谢物，多种细胞因子和趋化因子20,21,22和释放。新合成的产品发布时的独立SG发布。总的来说，这些调解员开展早期和晚期炎症和过敏性反应。因此，了解占MC激活和脱颗粒的机制是理论和临床小鬼ortance。 转基因操纵初级和培养管委会的困难阻碍了试图阐明基本MC脱颗粒的机制，仍然解决不好。为了克服这个问题，我们开发了一种基于记者通过测定共转染的粘膜肥大细胞系，大鼠嗜碱性白血病（RBL）-2H3（本文中称为RBL）或骨髓衍生的MC（的BMMCs）30与感兴趣的基因和神经肽Y（NPY）融合到单体RFP（MRFP），作为SG记者。 NPY先前所示扼要重述在其它系统的内源性SG标志物的行为。此外，由于荧光MRFP是pH值不敏感的，表达NPY-MRFP允许酸性的SG的可视化以及胞吐作用的定量评估通过使用96孔板和荧光板读数器。我们已经表明，NPY-MRFP被输送到RBL细胞和的BMMCs的酸性SGS和从该细胞释放在经调节的方式沿着内源性SG货物（ 即 ，β氨基己糖苷酶和血清素）30，32。这个协议提供了高分辨率成像为基础的方法，它允许对他们的表型和在RBL细胞32上SG上的特点和脱粒功能影响兴趣基因的筛选。具体地，该协议允许的MC的SG的实时跟踪和它们的面积或体积的大小，它们的数量，组装的动力学，其沿着细胞骨架的运动以及它们在不同条件下质膜最终融合的定量。例如，用DNP特异性IgE致敏的细胞和触发细胞用多价抗原（DNP缀合的血清白蛋白）根据不同的扰动（ 即 ，击倒的感兴趣的基因，过的重量或突变基因的表达，或药理学操作），并比较，以控制细胞。

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6046-500ML</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum</td><td>GE health care Life sciences</td><td>SH30071.01</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life technologies</td><td></td><td></td></tr><tr><td>Cellulose acetate membrane, pore size 0.22&amp;nbsp;&amp;mu;m</td><td>Sigma-Aldrich</td><td>CLS430769-1EA</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Trypsin/EDTA Solution (TE)</td><td>Life technologies</td><td>R001100</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>PIPES dipotassium salt</td><td>Sigma-Aldrich</td><td>108321-27-3&amp;nbsp;</td><td></td></tr><tr><td>Calcium acetate hydrate</td><td>Sigma-Aldrich</td><td>114460-21-8</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate</td><td>Sigma-Aldrich</td><td>M5661&amp;nbsp;</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate (Potassium glutamate)</td><td>Sigma-Aldrich</td><td>G1501</td><td></td></tr><tr><td>4 mm electroporation cuvettes</td><td>cell projects</td><td>EP-104</td><td></td></tr><tr><td>GENE PULSER WITH PULSE CONTROLLER &amp;amp; CAPACITANCE</td><td>Bio rad</td><td></td><td></td></tr><tr><td>Chambered coverglass</td><td>Thermo scientific</td><td>155411</td><td></td></tr><tr><td>24 well, flat bottom</td><td>Sigma-Aldrich</td><td>CLS3524</td><td></td></tr><tr><td>Corning 96 well plates</td><td>Sigma-Aldrich</td><td>CLS3367 or CLS390</td><td></td></tr><tr><td>96 well plate fluorescence reader- Infinite 200</td><td>Tecan</td><td></td><td></td></tr><tr><td>Calcium ionophore A23187</td><td>Sigma-Aldrich</td><td>C7522</td><td>Avoid from direct light exposure</td></tr><tr><td>12-O-tetradecanoyl-13-acetate (TPA)</td><td>Calbiochem</td><td>P3766</td><td></td></tr><tr><td>anti-DNP monoclonal IgE</td><td>Sigma-Aldrich</td><td>D8406&amp;nbsp;</td><td></td></tr><tr><td>DNP-BSA/ DNP-HAS</td><td>Sigma-Aldrich</td><td>A6661</td><td>Avoid from direct light exposure</td></tr><tr><td>Triton-x-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Confocal fluorescent microscope:</td><td></td><td></td><td></td></tr><tr><td>Zeiss LSM 510</td><td></td><td></td><td></td></tr><tr><td>Leica</td><td>SP5</td><td></td><td></td></tr><tr><td>Nikon A1 inverted</td><td></td><td></td><td></td></tr><tr><td>Imaris software</td><td>BITLANE</td><td></td><td></td></tr><tr><td>Microsoft exel or Prism or other analyses software</td><td></td><td></td><td></td></tr><tr><td>Other reagent:</td><td></td><td></td><td></td></tr><tr><td>Magnesium Chloride</td><td>MERK</td><td>5833</td><td></td></tr><tr><td>Sodium chloride</td><td>MERK</td><td>6404</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>MERK</td><td>2382</td><td></td></tr><tr><td>Bovine serum albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Glucose</td><td>BDH Laboratories</td><td>284515V</td><td></td></tr><tr><td>Monosodium phosphate&amp;nbsp;</td><td>MERK</td><td>5345</td><td></td></tr><tr><td>Sterile water</td><td></td><td></td><td></td></tr></tbody></table>

investigating mast cell secretory granules; from biosynthesis to exocytosis

肥大细胞（MC）是免疫系统的分泌细胞完成其生理和病理功能通过释放预先形成和新合成的过敏性，炎性和免疫调节介质。 MC的中介影响多个组织和器官最终在过敏和免疫反应。在MC介质的合成，储存和释放受到严格的监管。预形成的介质都装在胞质分泌粒剂（SG），该融合与质膜和由​​稳压胞吐作用释放其内容。我们提出了一个协议的基础上，共表达的与一个报告基因的兴趣被靶向SGS和被释放的调节的方式沿着内源性SG介质的基因。该协议使高分辨率四维共聚焦分析了MC的SGS和监控，从生源的时间表，以触发胞吐。因此，使用该协议除筛选基因EST他们的表型和功能的影响允许破译支配的MC的SG的生物合成和胞吐作用的分子机制，并确定所涉及的监管机构。由此，进一步的见解认为占在健康和疾病的MC功能的细胞机制应提供。

因为市政局的转染效率低的，遗传操作是不可能离开平均分泌的内源性的SG介质测量读数产生影响。然而，由在监测只表达感兴趣的基因的细胞群体建立完整的共表达的报告基因的NPY-MRFP和共转染质粒在相同细胞中，监测的NPY-MRFP结果。因此，这种测定法与传统方法相比的优点是能够选择性地监测遗传操作的细胞（ 图1）的能力。 事实上，采用此法，我们筛选基因?...

Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

本议定书的目标是开发一种方法，使肥大细胞分泌功能基因组分析。所述协议是基于荧光报道基因cotrasfected与感兴趣和实时的基因的释放定量评估分析分泌颗粒的形态。

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

investigating-mast-cell-secretory-granules-from-biosynthesis-to

Research

JoVE Journal

Immunology and Infection

13.1K 次观看.  Sackler Faculty of Medicine, Tel Aviv University. 本议定书的目标是开发一种方法，使肥大细胞分泌功能基因组分析。所述协议是基于荧光报道基因cotrasfected与感兴趣和实时的基因的释放定量评估分析分泌颗粒的形态。 

视频: Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

科研

免疫与感染

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

渗透胁迫对分泌性囊泡和胞吐作用的监测作用

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

神经科学

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

神经肽 Y-pHluorin 的共焦成像: 一种完整的小鼠和人胰岛胰岛素颗粒胞的可视化技术

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

生物学

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

成像 FITC-葡聚糖作为胞吐作用调节的记者

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

定量微模式细胞细胞外细胞异细胞的时态参数

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

Source: Tsvilovskyy, V., et al. <a href="https://app.jove.com/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.

研究目标化学品效果的肥大细胞脱颗粒测定

Source: Tsvilovskyy, V., et al. Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

Measuring Mast Cell Exocytosis via Neuropeptide Y Monomeric Red Fluorescent Protein Release

Source: Azouz, N. P., et al., <a href="https://app.jove.com/v/52505">Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis.</a> J. Vis. Exp. (2015)
This video demonstrates mast cell exocytosis induced by an activating reagent that includes a calcium ionophore, initiating the release of secretory granules containing the fluorescent reporter protein NPY-mRFP. Subsequently, we quantitatively assess the exocytosis by measuring the released NPY-mRFP using a fluorescence plate reader.

通过神经肽 Y 单体红色荧光蛋白释放测量肥大细胞胞吐作用

1. Measuring NPY-mRFP Exocytosis

	Preparation of Tyrode buffer:
	
		Prepare a solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in DDW. ...

Immunodiagnostics

Cellular Immunodiagnostics

An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

Source: Klein, O., et al. <a href="https://app.jove.com/v/57936">Imaging FITC-dextran as a Reporter for Regulated Exocytosis</a> J. Vis. Exp. (2018).
This video demonstrates a technique to visualize exocytosis in mast cells. The cells are incubated with FITC-dextran &#8212; a fluorescent reporter &#8212; which is taken up by cells inside secretory granules (SGs), where the acidic pH quenches its fluorescence. Upon stimulation of granule exocytosis, the medium&#39;s higher pH induces reporter fluorescence inside the fused SGs, which is visualized using a fluorescence microscope.

一种可视化肥大细胞中分泌颗粒胞吐作用的体外技术

1. Preparations

	Preparation of RBL (RBL-2H3 mast cell line) culture media
	
		Mix 500 mL of low glucose Dulbecco&#39;s modified Eagle&#39;s medium ...