Name: determination of the relative cell surface and total expression of recombinant ion channels using flow cytometry
Uploaded: 2016-09-28T15:00:00
Description: Watch this Scientific Journal Video about Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry at JoVE.com

We thank Mr. Serge S&#233;n&#233;chal and Dr. Jacques Thibodeau for sharing their expertise and granting us access to their flow cytometry and cell sorting platform. This work was completed with the operating grant 130256 from the Canadian Institutes of Health Research, a grant-in-aid from the Canadian Heart and Stroke Foundation, and support from the "Fondation de l'Institut de Cardiologie de Montr&#233;al" to L.P.

1.双标记的DNA构建<ol><li>通过定点诱变（ 图3B）20插入在D676和R677之间的Ca Vα2δ1的胞外接头的HA抗原决定基（YPYDVPDYA）。使用正向引物gccggattatgcgGGAAAACTCCAAACAACC和反向引acatcatacggataTCAATAAATTCATTGAAATTTAAAAGAAATTC。 </li><li>亚克隆被标记的HA的Ca Vα2δ1的cDNA序列为设计为表达融合到SacI和SalI位点（ 图3B）20之间mCherry的N-末端的蛋白质的哺乳动物pmCherry-N1?...

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此术为基础的流量测定成功地应用于电压门控钙通道14,22,26的荧光标记的成孔和相关的亚基的相对总和细胞表面水平的测量。调查基因突变的影响时，最好使用，因此要求该荧光标记标记的野生型构建体的固有荧光强度为至少10至100倍的荧光标记未标记的构建体的荧光强度大。在这个特殊的情况下，在此描述的mCherry钙Vα2δ1-HA构建体产生了较大的信噪比和更大的动态范围比对于先前...

本文提供了可靠的测定报告的膜蛋白如在使用现有的流式细胞仪技术的重组细胞中表达离子通道的相对细胞表面表达。离子通道是孔隙形成膜蛋白是负责通过门控离子穿过细胞膜的流控制的电信号。它们被激活的机制，性质和离子物质通过在那里它们被局部孔隙过渡的选择性分类。在细胞和组织水平，通过离子通道的宏观离子通量的生物物理（选通和渗透），生物化学（磷酸化），和生物合成（合成，糖基化，贩卖，和降解）特性1的产物。每个这些过程是唯一的每一种类型的离子通道，并且被优化以满足离子通道的生理作用。因此，在改变这些微调过程中通过继承或遗传修饰，通常被称为“离子通道”，可能是有害的细胞稳态。需要强调的是在细胞表面传递的离子通道的“正确”的金额为细胞稳态关键的是非常重要的。即使是很小的增加（增益的功能）和小下降（失函数）的离子通道活性有可能引起过一辈子了严重的病变的可能性。在成熟的离子通道的细胞表面输送的缺陷是在许多离子通道，如囊性纤维化（CFTR离子通道）2和长QT综合征形式（心脏钾通道）3的心律失常的重要决定因素。 离子通道与心脏猝死4有关。所有心脏离子通道的当前世界范围内流行被认为是至少为1：2,000-1：3000每个人5和负责一半左右突然心律失常心脏病死亡的CaSES 6。功能障碍在心脏的电压门控钠 - ，钾和钙 - 选择性离子通道已知在此过程中的关键作用。的L-型Ca V 1.2电压门控钙通道，需要发起同步心脏肌肉收缩。心脏L-型Ca V 1.2通道是α2δ1辅助亚基7-12的主孔形成的Ca Vα1亚单位和Ca V SS和Ca 的V组成的多亚基蛋白复合物。需要注意的是辅亚基的满装需要产生在质膜官能的Ca V 1.2通道和动态相互作用之间的这些亚基是必不可少的，以支持心脏13的正常电动功能。钙V SS促进钙Ⅴ的细胞表面表达通过非共价纳摩尔疏水相互作用14 1.2通道。钙Vα2δ1亚基无线的共表达次的Ca V SS结合的Ca Vα1刺激峰值电流表达式（5至10倍），并促进信道激活的更多负电压。增益的函数的孔隙形成亚基的Ca V 1.2已经用称为而形成L-型Ca V 1.2通道的三个主要亚单位的宿主点突变的长QT综合征15的室性心律失常的形式相关联的基因突变从短QT综合征形式16,17心律失常的受试者已经确定。离子通道是可以从生物化学观点（蛋白质化学）进行调查，或使用电的工具（电流产生的机器），并常常使用这些互补的方法膜蛋白。电生理学，特别是全细胞膜片钳，是阐明离子通道15的功能的合适的方法，但不能从它们的生物物理变化解决蛋白质运输的修改属性。蛋白质化学已然而，经常使用有限的，由于相对于较小的可溶性蛋白大的膜蛋白的相对低的表达。用荧光读数鲁棒高通量方法需要为了专门解决在蛋白质生物合成引起离子通道的细胞表面表达的变化的缺陷被开发。 流式细胞术是在细胞计数，分拣，生物标志物的检测，以及蛋白质工程18中采用的生物物理技术。当活细胞或颗粒的样品溶液被注入到流式细胞仪，细胞被排序成可由机器的检测系统（ 图1）被探测的单个流。第一流式细胞仪1956 19产生仪器检测到只有一个参数，但现代的流式细胞仪具有多个激光器和荧光检测器，使超过30荧光参数20,21的检测。镜和反射镜（光学发射）指示细胞的光散射或荧光到按比例转换成光的强度的电子网络（光电二极管和探测器）。数字数据使用专门的软件分析和主输出显示为一个点图21。 <img alt="图1" src="/files/ftp_upload/54732/54732fig1.jpg" /> 图1：流量生物物理学原理术分选的单细胞被通过喷嘴在高压下鞘流体的流，其通过一个或多个激光询问点移动他们内推。的光束被传递细胞偏转并在向前的方向（前向散射，FCS）的收集的光被发送到的光转换成成比例的小区的大小的信号的光电二极管。的光也收集在一个90°角的激光路径并传送到检测器（也称为光电倍增管（PMT））。如果激发荧光染料是存在于细胞中这个光透过分色镜允许侧向散射信号（SSC），这反映了细胞内的粒度的检测，和荧光发射路由。三个检测器（绿色，黄色和红色）被表示具有不同波长的带通滤波器，允许不同的荧光染料的同时检测。不同的信号被外部的计算机数字化并转换成将要分析量化细胞的特性数据。 <a href="http://ecsource.jove.com/files/ftp_upload/54732/54732fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 流式细胞仪的高通量容量利用来量化重组野生型和贩卖缺陷电压门控L-型Ca V 1.2通道和在活细胞相关亚基的相对膜表达。 cDNA构建合作丁对蛋白双重标记同时进行，可以由一个不渗透的荧光缀合的抗体和细胞内的荧光团是组成型荧光来检测细胞外的非荧光的表位。两者胞外表位，插在该蛋白质的细胞外环，与细胞内荧光团，C末端后插入，被翻译的蛋白质。在这一系列的实验中，钙Vα2δ1蛋白质工程改造以表达一种细胞外血凝素（HA）表位（YPYDVPDYA）由一个不渗透的FITC（异硫氰酸荧光素）检测到的缀合的抗HA和mCherry作为固有细胞内荧光团。为了确定mCherry钙Vα2δ1HA标记的蛋白的相对细胞表面的表达水平，表达的融合蛋白的重组细胞转染后，收获，并用FITC-缀合的小鼠单克隆抗HA表位标签antibod染色Y（ 图2）。 FITC是有机荧光化合物为比酶记者相当小，因此不作为可能与生物功能干涉。 mCherry-钙Vα2δ1-HA在TSA-201cells过表达时，产生在FITC荧光和mCherry荧光二维图22显著3-日志增加。鉴于该HA抗原决定基位于所述蛋白的细胞外部分，在完整细胞的存在下获得的FITC的荧光强度反映的HA标记的蛋白的细胞表面表达的相对指数。在结构的HA抗原决定基的可访问性系统通过测量细胞透后FITC信号验证。这项措施也有助于证实了标准化总蛋白的表达，因为对FITC荧光的相对强度在透细胞估计是定性相媲美的相对荧光值FOřmCherry透和非透化条件22,23下进行测定。要注意的是本征荧光光谱透后向更高值移位但相比于对照构建所报告的唯一值是在荧光强度的变化是很重要的。在荧光强度为测试构建体的相对变化所使用的ΔMean荧光强度（ΔMFI）值对于每个荧光团（mCherry或FITC）估算。实验设计用于测量相对于在相同条件下表达的对照构建的荧光强度测试构建体的荧光强度，以限制在所述荧光团共轭抗体的固有荧光实验的变化。使用该测定两膜蛋白被成功地研究了：在一个不同系列的L型电压门控钙通道的Ca V 1.2 14,22和孔形成亚基实验中，细胞外辅助的Ca Vα2δ1亚基22,23。以下方案用于测定1.2信道控制的条件下，影响离子通道的翻译后修饰的突变后的心脏L-型CaⅤ的钙Vα2δ1亚基的细胞表面表达。根据标准化的实验条件下，FITC的细胞表面的荧光增加的准线性的cDNA编码的mCherry钙Vα2δ1-HA蛋白质（ 图5从参考22）的表达。 <img alt="图2" src="/files/ftp_upload/54732/54732fig2.jpg" /> 图2：在流式细胞术实验方案的总流量和膜标签的示意图的方案概述了一些必要的主要步骤来量化由佛罗里达州的重组离子通道的相对总和细胞表面表达流式细胞仪。细胞在TSA-201细胞（1）用双标记的结构mCherry钙Vα2δ1-HA转染的和之前或透化后染色的（2）。多参数数据在流动收购仪（3）多因素分析（4）。 <a href="http://ecsource.jove.com/files/ftp_upload/54732/54732fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="763">
	<colgroup>
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	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td style="width:143px;">New England Biolabs</td>
			<td style="width:123px;">E0554S</td>
			<td style="width:281px;">Can be substitute with QuickChange site-directed mutagenesis Kit (Agilent, #200523).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubes 1,5 mL</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">72-690-001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubes 15 mL&#160;</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">&#160;62-554-002</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable graduated Tranfer Pipets&#160;</td>
			<td style="width:143px;">VWR</td>
			<td style="width:123px;">160001-192&#160;</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100-mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:123px;">430167</td>
			<td style="width:281px;">For standard culture of HEKT cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35-mm culture dish</td>
			<td style="width:143px;">Falcon</td>
			<td style="width:123px;">353001</td>
			<td style="width:281px;">For standard culture of HEKT cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 1 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1251.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 5 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1253.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 10 ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1254.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serological pipette 25ml</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">86.1285.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dulbecco&#39;s high-glucose medium</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">12100-046</td>
			<td style="width:281px;">Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fetal Bovine Serum, qualified, heat inactivated, US origin</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">16140-071</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">15140-122</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Lipofectamine 2000&#160;</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">11668-019</td>
			<td style="width:281px;">For liposomal transfection. Can be substituted with calcium phosphate transfection.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">31985-070</td>
			<td style="width:281px;">Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Trypsin-EDTA (1X) 0.05%, phenol red</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:123px;">25300-062</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microtubes</td>
			<td style="width:143px;">Sarstedt</td>
			<td style="width:123px;">72.690.001</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Phosphate Buffered Saline 1X</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">BP661-10</td>
			<td style="width:281px;">Can be &#34;home-made&#34;.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anti-HA FITC conjugated antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:123px;">H7411</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">IgG1&#8722;FITC Isotype Control antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:123px;">F6397</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="20">
			<td height="61" rowspan="2" style="height:61px;width:216px;">BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:123px;">554714</td>
			<td style="width:281px;">Fixation/Permeabilization. Permeabilization/Wash solution, store at 4 &#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:143px;"></td>
			<td style="width:123px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">49105161</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">15250061</td>
			<td style="width:281px;">To access cell viability.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Refrigerated Microcentrifuge, 5430R</td>
			<td style="width:143px;">Eppendorf&#160;</td>
			<td style="width:123px;">A14H172200</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Forma Steri-Cycle CO2 Incubator</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">370</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laboratory Platform Rocker</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:123px;">545034</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:143px;">VWR</td>
			<td style="width:123px;">89032-216</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSARIA III&#160;</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:123px;">648282</td>
			<td style="width:281px;">Flow cytometer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FlowJo Software v10</td>
			<td style="width:143px;">FlowJo</td>
			<td style="width:123px;">FlowJo v10 Dongle</td>
			<td style="width:281px;">For data analysis.</td>
		</tr>
	</tbody>
</table>

determination of the relative cell surface and total expression of recombinant ion channels using flow cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

本文介绍了一种可靠的协议通过双色流式细胞术测定法来量化TSA-201cells表达的重组的离子通道的总和细胞表面。作为一个例子，相对的细胞表面和总蛋白表达的钙Vα2δ1subunit定量。为了进行流式细胞术测定中两色流动，钙Vα2δ1被双重标记，以表示可被一个抗HA FITC缀合的抗体和组成型荧光灯内mCherry荧光团来检测一个细胞外的非荧光的表位的HA。对FITC和mCherry...

Watch this Scientific Journal Video about Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry at JoVE.com

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

继承心律失常通常是由改变一种或多种离子通道的表面上输送的突变引起的。在这里，我们适应流式细胞测定法，以提供在TSA-201细胞中表达的重组的离子通道的相对总和细胞表面蛋白表达的定量。

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Research

JoVE Journal

Biology

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Flow Cytometry Purification of Mouse Meiotic Cells

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...