Name: investigating mast cell secretory granules; from biosynthesis to exocytosis
Uploaded: 2015-01-26T14:00:00
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...

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W., Read, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agents+that+release+histamine+from+mast+cells.">Agents that release histamine from mast cells.</a> Annu Rev Pharmacol Toxicol. 23, 331-351 (1983).</li><li>Depinay, N., Hacini, F., Beghdadi, W., Peronet, R., Mecheri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-dependent+down-regulation+of+antigen-specific+immune+responses+by+mosquito+bites.">Mast cell-dependent down-regulation of antigen-specific immune responses by mosquito bites.</a> J Immunol. 176 (7), 4141-4146 (2006).</li><li>Abel, J., 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=Staphylococcus+aureus+evades+the+extracellular+antimicrobial+activity+of+mast+cells+by+promoting+its+own+uptake.">Staphylococcus aureus evades the extracellular antimicrobial activity of mast cells by promoting its own uptake.</a> J Innate Immun. 3 (5), 495-507 (2011).</li><li>Avila, M., Gonzalez-Espinosa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+Toll-like+receptor+4+and+mast+cell-dependent+innate+immunity+responses.">Signaling through Toll-like receptor 4 and mast cell-dependent innate immunity responses.</a> IUBMB Life. 63 (10), 873-880 (2011).</li><li>Novak, N., Bieber, T., Peng, W. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+proteases+as+protective+and+inflammatory+mediators.">Mast cell proteases as protective and inflammatory mediators.</a> Adv Exp Med Biol. 716, 212-234 (2011).</li><li>Lundequist, A., Pejler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+preformed+mast+cell+mediators.">Biological implications of preformed mast cell mediators.</a> Cell Mol Life Sci. 68 (6), 965-975 (2011).</li><li>Metz, M., Maurer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells--key+effector+cells+in+immune+responses.">Mast cells--key effector cells in immune responses.</a> Trends Immunol. 28 (5), 234-241 (2007).</li><li>Gordon, J. R., Burd, P. 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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>

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。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Name of Material/ Equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:156px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:421px;">DMEM</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D6046-500ML</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fetal Bovine Serum</td>
			<td style="width:128px;">GE health care Life sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td style="width:128px;">Life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Cellulose acetate membrane, pore size 0.22&#160;&#956;m</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning tissue-culture treated culture dishes</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:128px;">Life technologies</td>
			<td>R001100</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">PIPES dipotassium salt</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">108321-27-3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium acetate hydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>114460-21-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium acetate tetrahydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">M5661&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">L-Glutamic acid potassium salt monohydrate (Potassium glutamate)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 mm electroporation cuvettes</td>
			<td style="width:128px;">cell projects</td>
			<td style="width:139px;">EP-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GENE PULSER WITH PULSE CONTROLLER &#38; CAPACITANCE</td>
			<td style="width:128px;">Bio rad</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chambered coverglass</td>
			<td style="width:128px;">Thermo scientific</td>
			<td align="right" style="width:139px;">155411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">24 well, flat bottom</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Corning 96 well plates</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3367 or CLS390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate fluorescence reader- Infinite 200</td>
			<td style="width:128px;">Tecan</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium ionophore A23187</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">C7522</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-O-tetradecanoyl-13-acetate (TPA)</td>
			<td>Calbiochem</td>
			<td height="20" style="height:20px;width:139px;">P3766</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-DNP monoclonal IgE</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D8406&#160;</td>
			<td height="20" style="height:20px;width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNP-BSA/ DNP-HAS</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A6661</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Triton-x-100</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Confocal fluorescent microscope:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Zeiss LSM 510</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Leica</td>
			<td style="width:128px;">SP5</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon A1 inverted</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Imaris software</td>
			<td style="width:128px;">BITLANE</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Microsoft exel or Prism or other analyses software</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Other reagent:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium Chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5833</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sodium chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">6404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium chloride&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">2382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A4503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Glucose</td>
			<td style="width:128px;">BDH Laboratories</td>
			<td style="width:139px;">284515V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monosodium phosphate&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sterile water</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></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 ...

Research

JoVE Journal

Immunology and Infection

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

科研

免疫与感染

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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

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 ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

使用"桅杆细胞敲击"小鼠分析体内桅杆细胞的功能

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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

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

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 ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

通过使用来自辐照的Buffy外套的饲养细胞的T淋巴细胞的分离和培养来研究主动脉瓣钙化

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...