Name: isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays
Uploaded: 2018-07-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays at JoVE.com

作者想确认朱莉娅 Geminn 在肥大细胞的制备和培养方面的技术援助。这项工作得到了跨区协作研究中心 (SFB) 152 的支持。

所有动物规程根据德国立法指南被执行了对实验室动物的关心和用途 (正式地批准由卡尔斯鲁厄区域理事会)。
1. 经腹腔灌洗的 PMC 分离和培养
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			<td>冰</td>
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<td>2</td>
			<td>10毫升注射器</td>
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<td>3</td>
			<td>27克针</td>
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<td>4</td>
			<td>20克针</td>
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<td>5</td>
			<td>泡沫塑料块和别针</td>
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<td>6</td>
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<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J Biol Chem. 260 (6), 3440-3450 (1985).</li><li>Schwartz, L. B., Austen, K. F., Wasserman, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+release+of+beta-hexosaminidase+and+beta-glucuronidase+from+purified+rat+serosal+mast+cells.">Immunologic release of beta-hexosaminidase and beta-glucuronidase from purified rat serosal mast cells.</a> J Immunol. 123 (4), 1445-1450 (1979).</li><li>Schafer, B., 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=Mast+cell+anaphylatoxin+receptor+expression+can+enhance+IgE-dependent+skin+inflammation+in+mice.">Mast cell anaphylatoxin receptor expression can enhance IgE-dependent skin inflammation in mice.</a> J Allergy Clin Immunol. 131 (2), 541-549 (2013).</li><li>Maurer, M., 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=Mast+cells+promote+homeostasis+by+limiting+endothelin-1-induced+toxicity.">Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.</a> Nature. 432 (7016), 512-516 (2004).</li><li>Maurer, M., Church, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+skin+responses+induced+by+icatibant+injection+are+mast+cell+mediated+and+attenuated+by+H(1)-antihistamines.">Inflammatory skin responses induced by icatibant injection are mast cell mediated and attenuated by H(1)-antihistamines.</a> Exp Dermatol. 21 (1), 154-155 (2012).</li><li>McNeil, B. D., 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=Identification+of+a+mast-cell-specific+receptor+crucial+for+pseudo-allergic+drug+reactions.">Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.</a> Nature. 519 (7542), 237-241 (2015).</li><li>Gaudenzio, N., 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=Different+activation+signals+induce+distinct+mast+cell+degranulation+strategies.">Different activation signals induce distinct mast cell degranulation strategies.</a> J Clin Invest. 126 (10), 3981-3998 (2016).</li><li>Solis-Lopez, A., 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=Analysis+of+TRPV+channel+activation+by+stimulation+of+FCepsilonRI+and+MRGPR+receptors+in+mouse+peritoneal+mast+cells.">Analysis of TRPV channel activation by stimulation of FCepsilonRI and MRGPR receptors in mouse peritoneal mast cells.</a> PLoS One. 12 (2), 0171366 (2017).</li><li>Enerback, L., Svensson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+peritoneal+mast+cells+by+centrifugation+on+density+gradients+of+Percoll.">Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll.</a> J Immunol Methods. 39 (1-2), 135-145 (1980).</li><li>Bird, G. S., DeHaven, W. I., Smyth, J. T., Putney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+store-operated+calcium+entry.">Methods for studying store-operated calcium entry.</a> Methods. 46 (3), 204-212 (2008).</li><li>Aronson, N. N., Kuranda, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+degradation+of+Asn-linked+glycoproteins.">Lysosomal degradation of Asn-linked glycoproteins.</a> FASEB J. 3 (14), 2615-2622 (1989).</li><li>Moon, T. C., Befus, A. 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mc 模型可以成功地用于研究 mc 脱粒, 趋化性, 黏附性, 以及阐明细胞内信号传导通路参与 mc 激活。一些研究人员使用人体 mcs 模型, 如永生化的线 (LAD2 和 HMC1) 或由脐血祖细胞 (CD133+) 和外周血祖细胞 (CD34+) 所衍生的人 mcs。其他人更喜欢啮齿动物 MC 模型, 如永生化 (大鼠嗜碱性颗粒白血病 RBL-2H3 MC 线) 或初级培养 (小鼠骨髓源性 MCs BMMCs, 局) 模型。小鼠原发性 MCs 可用于分析多种 "敲出" 老鼠模型中的 MC ...

MCs 在先天和后天免疫应答中起着突出的作用。具体地说, MCs 参与杀害病原体, 如细菌和寄生虫, 并降低潜在的有毒内源性肽或毒液成分 (审查见加利等20081)。MCs 在先天和适应性免疫中的生理作用是激烈争论的主题。因此, 在不同的 MC 缺陷小鼠模型的研究中, 大量的数据差异需要对2过敏后的 MCs 免疫功能进行系统的重新评估。成熟的 MCs 大多是局部的组织和器官, 如皮肤, 肺和肠道, 通常只在少量的血液中发现。MCs 来源于造血前体, 如 MC 祖细胞, 在几乎所有血管化组织的微环境中完成它们的分化和成熟1。T 细胞衍生因子白细胞介素 (IL)-3 有选择地促进小鼠 MCs 的多能种群的生存能力, 增殖和分化从他们的造血祖父母3。干细胞因子 (超临界) 是由组织中的结构细胞产生的, 在 MC 的发展、生存、迁移和功能4中起着至关重要的作用。单个 MCs 的性质可能因其合成和贮存各种蛋白酶或软骨的能力而异。在小鼠中, 根据肝素和蛋白酶5的解剖定位、形态学和含量, 将所谓的结缔组织型 mcs 与黏膜 mcs 区别开来。
在 MCs 中, 增加游离胞浆 Ca2 + ([Ca2 +]i) 是脱粒和生产 eicosanoids, 以及细胞因子的合成和转录因子的活化反应的抗原和各种 secretagogues6。这些刺激的主要下游目标是磷脂酶 C, 水解磷酸肌醇 45-bisphosphate 到甘油 (达格·哈马舍尔德) 和肌醇 14, 5-trisphosphate。IP3 激活蛋白激酶 C, 并从内质网中释放出钙2 +的离子。这些商店的枯竭激活了 ca2 +通过等离子膜 ca2 +通道流入, 导致储存操作钙进入 (SOCE)。这一过程是通过与 Ca2 +传感器, 基质相互作用 molecule-1 (Stim1) 的互动, 在内质网与 Orai17以及通过活化的瞬态受体电位规范 (TRPC) 通道蛋白质 (复习见 Freichel等。20148) 在血浆膜。
为了研究这些通道的生理作用, 通常使用几种药理学 (通道阻滞剂的应用) 或遗传方法。在后一种情况下, 抑制蛋白质表达是通过靶向 mRNA (击倒) 或基因组 DNA 编辑与全球或组织特定的9删除一个外显子编码一个孔形成亚单位的通道 (挖空)。对这些通道有足够特异性的拦截剂的供应是有限的。此外, 被击倒的方法需要仔细控制它的有效性使用, e., 西方污点分析, 并由于缺乏特定的抗体为靶向的渠道蛋白在许多情况下。因此, 剔除鼠标模型的使用仍然被认为是这种分析的黄金标准。对 MC 功能进行调查的一种优选的体外模型是培养可被隔离为完全成熟的种群 (相对于体外, e., 骨髓细胞的不同的 MCs)10.与骨髓源性 MCs (BMMCs) 相比, 它也通常用于研究 MC 功能的体外, FcεRI 和β hexosaminidase 释放的刺激分别增加8倍和100倍。在本文中, 我们描述了一种分离和培养小鼠的方法, 它允许在2周后获得大量的纯结缔组织类型的 MCs, 足以进一步分析。
尽管最近在开发和应用基因编码钙指标方面取得了进展, 但它们的使用仍受限于向目标细胞提供基因的困难, 特别是在诸如初级培养的 MCs 等高度专门化的细胞中。此外, 这组荧光指示器为 [2 +]i测量仍然缺乏比例染料以一个高动态范围。因此, 比例 [Ca2 +]i测量的首选方法仍然是使用荧光染料 "Fura-2"11。
目前, 对 MC 活化和脱粒的评价最常用的方法是β-hexosaminidase 活性的测量。β-hexosaminidase, 过敏性调解人, 是 MC 颗粒成分之一, 与组胺的联合释放, 从 MCs12的恒定比例。β-hexosaminidase 可以通过其与特定基质的反应而容易而准确地测量, 从而产生可测量的 colorigenic 产品的数量, 在微板块比色法中很容易检测到。本文介绍了该技术在脱粒分析中对各种刺激反应的应用。
高亲和 plasmalemmal IgE 受体 (FcɛRI) 的聚集在 MCs 中激活一个多功能的细胞内信号通路, 导致分泌颗粒含量在周围细胞外空间释放。除了特定的抗原, 许多其他刺激可以激活 MCs 释放多种免疫调节介质, 如补充 anaphylatoxins (e., C3a 和 C5a)13, 收缩肽内皮素 1 (ET1)14, 以及许多阳离子物质和药物引起的 pseudoallergic 反应 (e., icatibant)15通过绑定到 MRGPRX216。MRGPR 诱导的 MCs 脱粒细胞内信号通路与 FcɛRI 介导的细胞内信号传递的特征相比较差;这些途径只开始密集地被研究在最近几年17在受体识别16以后。在很大程度上, 涉及钙进入 MRGPRX2 刺激的等离子膜离子通道仍有待了解。因此, 本文还重点研究了 MRGPR 激动剂刺激的 MCs 细胞内钙信号和脱粒。

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">RPMI Medium</td>
			<td style="width:273px;">Thermo Scientific</td>
			<td style="width:212px;">21875034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPBS</td>
			<td>Sigma-Aldrich</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Thermo Scientific</td>
			<td>R92157</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IL-3</td>
			<td>R&#38;D Systems</td>
			<td>403-ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SCF</td>
			<td>Thermo Scientific</td>
			<td>PMC2115</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Concanavalin A</td>
			<td>Sigma-Aldrich</td>
			<td>C2272</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fura-2 AM&#160;</td>
			<td>Thermo Fischer Scientific</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNP-HSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6661</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-DNP-Antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D-8406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penstrep</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compound 48/80&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C2313</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">4-Nitrophenyl 2-acetamido-2-deoxy-&#946;-D-glucopyranoside</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">N9376</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CoverWell Imaging Chamber</td>
			<td>Sigma-Aldrich</td>
			<td>GBL635051</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96-Well plate, v-shaped bottom&#160;</td>
			<td>Corning</td>
			<td>3896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96-Well plates, flat bottom</td>
			<td>Greiner Bio-One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microtiter plate reader with &#34;i-control&#34; software&#160;</td>
			<td>Tecan</td>
			<td>Nano Quant, infinite M200 Pro</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flat bottom plate&#160;</td>
			<td style="width:273px;">Greiner Bio-One</td>
			<td style="width:212px;">655180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">I9657</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50 mL Plastic Centrifuge Tubes</td>
			<td>Sarstedt</td>
			<td style="width:212px;">62.547.254&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Culture Flasks (25 cm)</td>
			<td style="width:273px;">Greiner Bio-One</td>
			<td>69160</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cover slips glasses round; &#248; 25 mm; No. 1</td>
			<td>Menzel</td>
			<td>CB00250RA1</td>
			<td></td>
		</tr>
		<tr height="24">
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			<td>631-0696</td>
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		<tr height="24">
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			<td>Zeiss</td>
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			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;">Objectiv Fluar 40x/1.3 Oil</td>
			<td>Zeiss</td>
			<td style="width:212px;">440260-9900</td>
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			<td height="24" style="height:24px;">HC Filter Set Fura 2&#160;</td>
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			<td>Zeiss</td>
			<td>AxioCam MRm&#160;</td>
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		<tr height="24">
			<td height="24" style="height:24px;">Monochromatic Light Source</td>
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			<td>Lambda DG-4</td>
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		</tr>
		<tr height="24">
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			<td>Zeiss</td>
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		<tr height="24">
			<td height="24" style="height:24px;">Gravity fed solution application system</td>
			<td>Custom Made</td>
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		<tr height="24">
			<td height="24" style="height:24px;">15 mL Plastic Centrifuge Tubes</td>
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			<td>62,554,502</td>
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		<tr height="24">
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			<td style="width:212px;">Megafuge 1.0R</td>
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isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays

肥大细胞 (MCs) 作为免疫系统的一部分, 在保护宿主抵抗几种病原体和引发过敏性免疫应答方面起着关键作用。通过将表面 IgE 与高亲和 IgE 受体 (FcεRI) 的交联, 以及通过其他几种受体的刺激, 激活 MCs, 启动了游离胞内 Ca2 +级 ([Ca2 +]i) 的上升。这促进了炎症和过敏介质的释放。这些信号通路中所涉及的分子成分的识别对于理解 MC 函数的调节是至关重要的。本文介绍了一种通过腹腔灌洗和腹腔 MCs 培养分离小鼠结缔组织型 mcs 的协议。采用这种方法对各种击倒小鼠模型的 MCs 进行培养, 是鉴别 MC 信号通路所涉及蛋白质的一种有用方法。此外, 我们还描述了一个单细胞 Fura-2 成像协议, 作为一个重要的技术, 以量化的 Ca2 +信号在 MCs. 基于荧光的监测 [ca2 +]我是一个可靠和常用的方法来研究 Ca2 +信号事件, 包括存储操作的钙进入, 这是至关重要的 MC 激活。对于 MC 脱粒的分析, 我们描述了β hexosaminidase 释放试验。将β-hexosaminidase 释放到培养基中的量被认为是 MCs 中描述的所有三种不同分泌亚群的脱粒标记. β-hexosaminidase 可以很容易地被量化的反应与 colorigenic 基板在微量滴定板比色法。这种高度可重复的技术具有成本效益, 不需要专门的设备。总体而言, 所提供的协议表明了一个高产量的 mcs 表达典型的 MC 表面标记, 显示典型的形态和表型特征的 mcs, 并表明高重现性反应 secretagogues 在 Ca2 +-影像学和脱粒化验。

在无菌湿润条件下, 经腹腔灌洗并进一步培养 RPMI 培养基中的3种野生小鼠 (C57BL/6N), 在生长因子 (IL-3 10 ng/毫升和 30 ng/毫升) 的情况下, 对其进行了进一步养殖。37°c 和 5% CO2。媒介在天2和9被改变了在细胞隔离以后。用透射光 DIC 成像 (见图 1A, B) 对细胞形态学进行分析。细胞的对比度和粒度表现出良好的细胞活力。在这些条件下培?...

Watch this Scientific Journal Video about Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays at JoVE.com

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

在这里, 我们提出了一个隔离和培养小鼠腹膜肥大细胞的协议。我们还描述了两个协议的功能表征: 一个荧光成像的细胞内游离 Ca2 +浓度和脱粒检测的基础上的色度量化的释放β-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 ...

Research

JoVE Journal

Immunology and Infection

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

Isolation of Functional Cardiac Immune Cells

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

科研

免疫与感染

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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

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 ...

Peptide-based Identification of Functional Motifs and their Binding Partners

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...