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表达载体。 注意:适当的通道功能需要使用标准的电生理学方法24的控制结构进行测试。 </li></ol> 2.脂质体介导瞬时转染（30分钟，所有的步骤中进行在层流罩） <ol><li>第1天:板五十万TSA-201细胞（或HEKT）在35毫米培养皿用Dulbecco的2毫升的高葡萄糖的最低必需培养基（DMEM-HG）补充有10％胎牛血清（FBS）和1％青霉素 - 链霉素（PS）的培养基中。算使用标准血细胞计数器细胞。使用台盼蓝的细胞样品的一小部分评估细胞生存力。板足够的细胞在转染时达到90％汇合。 </li><li>第2天:用2ml新鲜预热（37℃）培养液的无PS变化的培养基。 </li><li>对于每一个转染样本，准备2个1.5 mL管。在管1，稀4微克DNA在250微升血清培养基降低。在管2，混合10微升用250微升血清减少培养基中脂质体介导的转染试剂。使用前轻轻混合转染试剂。 </li><li>孵育在室温下5分钟。 </li><li>结合管1和管2的内容，轻轻混匀孵育在室温下至少20分钟。 </li><li>添加脂质体/ DN一个复合物培养的细胞，轻轻摇晃培养皿中混合。 </li><li>在37℃孵育下，5％CO 2气氛中 24小时。 </li></ol> 3.细胞的流式细胞染色（3小时） <ol><li>准备电池样品<ol><li>第3天:小心地取出从培养皿中并用400μl的预热（37℃）0.05％胰蛋白酶-1X EDTA（乙二胺四乙酸）洗涤细胞。 </li><li>添加400μl的胰蛋白酶-EDTA，并在5％的CO 2气氛中孵育在37℃下的菜5分钟以使细胞从培养皿中分离。 </li><li>通过加入1毫升冷的培养基中无PS停止酶消化并吹打轻轻4-5倍洗出从表面所有的细胞。避免过度消化和过度吸液，减少细胞死亡。 </li><li>收集细胞1.5毫升管，并立即置于冰上。使用冰冷的溶液，并保持细胞在4℃下，以防止内在化表面抗原。减少照明，以限制所述荧光信号的光漂白。 </li><li>离心管，在400×g离心在4℃下5分钟。小心吸弃上清。 </li><li>重悬在1ml磷酸盐缓冲盐水1X的粒料（PBS）中以制备单细胞悬浮液。 </li><li>简而言之轻轻涡管并重复步骤3.1.5和3.1.6彻底清除培养基。 </li><li>重悬在600微升1×PBS中的沉淀和调节细胞浓度至最小的3×10 6个细胞/ ml。 </li><li>分裂细胞有两种新的1.5毫升管的细胞外和细胞内染色。包括适当的控制，以区分与非特异性染色特异性染色。 注:同种型对照抗体有助于评估背景染色的水平和应该理想匹配每个初级抗体的宿主物种，同种型和荧光团。使用同种型对照和缀合的抗体在相同的蛋白质浓度。 </li></ol></li></ol><img alt="表格1" src="/files/ftp_upload/54732/54732table1.jpg" /> 表1: 流式细胞仪对于实验对照样品不透和透化的细胞的每个实验需要包括以下阴性对照:（1）未转染的细胞（无抗体，同种型或同偶联的抗体）。 （2）转染的细胞与感兴趣的质粒亚克隆而不构荧光细胞内荧光蛋白（pCMV-钙Vα2δ1-HA）或者与双重标记的结构（pmCherry钙Vα2δ1和无抗体温育，用同种型或同共轭抗体）。单一的色彩控制用于荧光发射重叠的赔偿。相同的控制将运行于非透化，并在各系列实验透条件。 <ol start="2"><li>的完整细胞表面染色活细胞<ol><li>等分试样1×10 6细胞/100μl的在1.5ml管中。 </li><li>添加FITC偶联的单克隆抗HA抗体以5微克/毫升和，涡流在4℃下孵育细胞上在黑暗中摇杆平台（200rpm）下进行45分钟前。 注:在初步滴定实验测定抗体的最佳浓度（ 图4）。 </li><li>从黑暗中取出细胞，并添加900微升1×PBS /管。离心机在400×g离心在4℃下5分钟。 </li><li>吸出上清液，并在400 xg离心重悬沉淀在1ml 1×PBS中，涡旋并离心在4℃下5分钟。 </li><li>重复洗（步骤3.2.4）两次以去除任何未结合的抗体。如果使用未缀合的初级抗体，孵育用适当的二级抗体。 </li><li>最后一次洗涤后，重悬细胞于500μl1×PBS中的和传送单细胞悬浮液在5毫升流式细胞术管。保持细胞S IN在4℃黑暗中直到运行样品。 </li><li>在流式细胞仪上运行样本。为了达到最佳效果，分析仪上尽快但不迟于24小时后流的单元格。 </li></ol></li><li>细胞内染色:固定，通透和染色<ol><li>等分试样1×10 6细胞/100μl的1.5毫升管和离心机在400×g离心在4℃下5分钟。 </li><li>弃上清，悬浮细胞在100微升直接从股票注视通透的解决方案。 </li><li>孵育在黑暗中在4℃下20分钟。 </li><li>添加新鲜配制的1X通透，洗涤液100微升（稀释蒸馏水2 O 10倍通透，洗涤液）。使用在400×g离心5分钟一台离心机在4℃涡流和沉积物的细胞。 </li><li>吸弃上清。 </li><li>重复步骤3.3.4和3.3.5。 </li><li>添加FITC偶联的单克隆抗HA抗体以5微克/毫升在100μl1×透洗涤缓冲液中，于4℃下30分钟在黑暗中温育细胞前涡流。 注意:下面的相同的步骤，用于细胞表面染色的一种是进行细胞内染色。皂素介导的细胞透然而，一个快速可逆的过程，因此，用1×彼尔姆/洗涤缓冲液，以取代1×PBS中，以保持细胞的皂素的细胞内染色过程中恒定存在是重要的。 </li><li>从黑暗中取出细胞，并加入100微升通透，洗涤液。离心机在400×g离心在4℃下5分钟。 </li><li>吸小心上清，重悬在100μl透洗涤缓冲液，涡旋和离心沉淀，在400×g离心在4℃下5分钟。 </li><li>重复洗（步骤3.3.9）1次，以去除任何未结合的抗体。 </li><li>最后一次洗涤后，重悬细胞于500μl1×PBS中的和传送罪GLE细胞悬液5毫升流式细胞仪管。保持在黑暗中的细胞在4℃下直到样品注入流式细胞仪。 </li><li>在流式细胞仪上运行样本。尽快但不迟超过1周染色后运行的流式细胞仪的固定样本。运行在同一天，非透化和透化的细胞。 </li></ol></li></ol> 4.流式细胞仪<ol><li>流式细胞仪细胞分选日报安装<ol><li>打开流式细胞仪软件。试验，标定和设置流式细胞仪细胞分选流程，以确保最佳的仪器性能之前使用的仪器设置珠（ 即激光和光学系统，满足规格，激光和流动池正确对齐）。 </li><li>使用100微米的喷嘴以20 PSI鞘压力。 注:该喷嘴不具有的长椅上改变流式细胞仪。 </li><li>根据厂家专业设置流式细胞仪的流率呃规范。非常高的流速将在检测的荧光变化的降低的敏感度。 </li><li>选择蓝色（488纳米激发荧光Isothiocayanate或FITC）和黄绿色（561纳米激发mCherry）激光器。收集FITC和mCherry荧光水平与530/30 nm和分别与二十零分之六百十纳米的带通滤波器。 </li><li>获得前向散射（FCS）对侧向角散射（SSC）的点积用线性刻度未染色细胞。调整各检测器的放大的可视化的细胞中的点图的左下象限。 </li></ol></li><li>完整的非渗透细胞的样品读数<ol><li>由划定游离形式围绕待分析的细胞不包括细胞碎片和细胞聚集体，从而限制了荧光信号以完整细胞设置P1gate活非透化的细胞。 注:活/死排斥染料可以用来促进活细胞上闸门布置。设置10,000个事件记录在止动门P1。设置此向更高一些事件如果需要的话。 </li><li>收购mCherry与FITC双参数等高线图来检测未染色细胞的自发荧光基线。使用双对数刻度显示负值，提高人口25之间的分辨率。调整各检测器的电压来设置所述第一10个单位的日志荧光强度曲线的下部分内的未染色的阴性细胞。 </li><li>通过收购成立于4.1.5和4.1.6设置都完好无透样本和收集FSC，SSC和信号的荧光检测器。 </li><li>出口和保存* .fcs文件被用于使用流式细胞分析软件分析。 </li></ol></li><li>透细胞样本解读<ol><li>移动至P1栅极透样品中选择活细胞和所示4.1.5和4.1.6调整FSC和SSC电压。 </li><li>收购全部透样本和收集FSC，SSC一在荧光检测第二信号。 </li><li>出口和保存* .fcs文件被用于使用流式细胞分析软件分析。 </li></ol></li><li>数据分析<ol><li>启动仪保存在4.2.4和4.3.3分析软件和进口* .fcs文件流。 </li><li>单击工作区窗口中列出的第一个样本。一个管ID号命名的新窗口自动打开。在SSC的与FSC的情节开始浇注过程。绘制周围使用活细胞的椭圆图标栅极（P1）和消除任何碎片，死细胞，或聚集体具有比活细胞不同的前向散射和侧向散射</li><li>绘制mCherry（y轴）对FITC（x轴）的活细胞的荧光强度的双参数等高线图，点击首先在x轴和选择的FITC的信道，然后单击在y轴并选择PE-mCherry-A通道。点击"四"图标象限标记处的边缘位置utofluorescent细胞各荧光通道。 注:门周围的FITC和mCherry阳性细胞集是P2大门。荧光阴性细胞群称为P3栅极。参见图5本文中所使用的典型门的方法。 </li><li>选择P2和P3盖茨和点击原工作区窗口中的"添加统计信息"图标。点击"计数"（阳性细胞数的），然后点击"中庸"（平均荧光每个荧光强度）或"中间"（每个荧光染料的荧光强度中值）的选项列表中的统计信息。再次单击"添加统计信息"图标。所有这些值都自动转移到原来的工作区窗口。 注:"平均"只有当荧光强度服从正态分布使用。在所有其他情况下，单击"中间"选项卡。因此，小额信贷机构可以参考平均荧光Intensity或中位数的荧光强度。 注:下一步骤是栅极&#39;参数和统计信息应用于通过流式细胞仪探测的所有样本。 </li><li>在工作区窗口中，用鼠标拖放盖茨和统计参数上标出所有样本。 </li><li>生成二维等值线图（mCherry VS FITC）和直方图（细胞计数与荧光强度）对非透化和透化的细胞（ 图6A - B）的批处理报告。 </li><li>从在步骤4.4.4生成的统计信息，计算每个荧光的染色的细胞的平均荧光强度（MFI）。从这个值，减去未染色的细胞获得的表面和感兴趣的蛋白质的总表达量化MFI值。 </li><li>报告各荧光（mCherry和FITC）（ 图6C - D）的ΔMFI值。规范化的钙测量的ΔMFI <suB&gt; Vα2δ1构建突变体，为FITC和mCherry与WT构建体获得的ΔMFI值。 注:荧光强度的绝对值可能大幅变化，这取决于批量的抗体和各实验室工作人员的技术能力，因此需要正常化使用WT构建体中的突变体构建体的荧光强度。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

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公布的Ca V 1.2-HA和Ca V 2.3-HA构建体测定的。这有很多原因可想而知。可以推测，HA抗原决定基中的钙的V＆的胞外域的本地环境＃945;2δ1蛋白质最大化信号到FITC偶联抗HA抗体的信噪比。 另外，它要记住，这种方法没有提供在细胞表面蛋白的数字的绝对值是重要的。可通过侧膜片钳进行侧和流式细胞仪的相同条件下的实验中所示的参考22 图7仍然获得，以获得功能性通道表达所需的标记构建体的相对表达。从这些数据，可以预计，峰值电流密度，离子通道活性的全局量度，通常随着标记的Ca Vα2δ1亚基的表面表达的功能。该电流细胞表面表达测定法的主要限制仍然是它不能在这个时候被应用到内源性离子通道的研究。这是由于这样的事实，即几个市售抗体已知特异性结合离子通道的预测外部域。因此它需要的蛋白质的一级序列的遗传操作在未分化重组细胞系进行研究表达如TSA-201细胞。 在测定的优化关键步骤:将适当对荧光团的鉴定，外部表位的选择和插入部位的定位是该方法的成功是至关重要的，因为所述表位的插入不应干扰蛋白质的功能。不同荧光团的组合进行了测试。两个外部表位:血凝素（HA）表位（YPYDVPDYA）和银环蛇毒素-结合位点的表位（WRYYESSLEPYPD）27和三个不可渗透的荧光标记的抗体进行评价，即FITC（异硫氰酸荧光素） -时，R-藻红蛋白- ，和在PE-Cy7-结合的抗体。此外，超过10个不同的插入网站外部HA抗原表位进行了钙Vα2δ1内进行测试。对于该表位的插入要求定点诱变和重组DNA技术，建议在同一区域内设计一个最小的三套引物。用9个残基，所述HA抗原决定基为其中存在市售的荧光标记的抗体，但在插入27个核苷酸的成功率比点突变下的小的表位中的一个。四半胱氨酸基序（残基半胱氨酸-半胱氨酸-Pro的基-Gly-半胱氨酸-半胱氨酸）比HA抗原决定基2个残基的长度，但荧光砷发夹粘合剂是膜透膜，不适合的膜结合蛋白28的分析。初步实验用两种细胞内的荧光团mCherry和绿色荧光蛋白（GFP）进行。三个标准被用来鉴别成功一对荧光团与离子通道:的荧光团需要1）发射光谱不重叠（详情请参阅参考资料20,21）; 2）标记的构建体的表达产生在如通过标准电生理学方法验证重组细胞功能的离子通道; 3）控制构建体的表达产生一个强大的荧光读数。如果上述条件之一不满足，例如，如果标签或荧光的插入与通道功能的干扰，一个人回到了起点和修改外部表位和/或插入位点的插入任一网站的荧光。该蛋白质的C-末端和荧光团之间的5谷氨酰胺残基的连接物可有助于改善蛋白功能。此外，它可能有助于插入在未已知在蛋白的功能和/蛋白质糖基化的关键角色大环路外部的表位。人们还需要注意的小的技术问题。 脂质体介导TRANSF的成功率挠度在TSA-201细胞能钙Vα2δ1内的两个表位的插入后降低。因此，建议重新校准与新的DNA构建体的转染效率。这是因为一个30 k​​Da蛋白的插入改变了DNA的分子量。如有必要，3或孵育将细胞在37℃下在较长的时间段（24-72小时）:一个可以修改的DNA的脂质体的比例为1:2至1。选择合适的制剂为透化细胞中，这是很重要的。评估该项目最自制的解决方案被证明导致细胞聚集和细胞仪堵塞的流动。 抑制光曝光而染色的荧光团共轭抗体的细胞是必不可少的，以限制光漂白和信号损失。为了最大限度地减少荧光背景，在使用前向离心过量缀合的抗体是很重要的。最后，这是在尽重要性来分析Ť他在细胞流式细胞仪尽快尽可能不迟于24小时后的流量。过夜保持染色的细胞在4℃下将有可能降低信号和损害细胞的存活。在批次的荧光团共轭抗体的固有荧光的变化决定了测试构建体的荧光强度需要作为对照构建体的荧光强度的函数相同的实验条件下表达进行报告。 以实现一个总体良好的信噪比，建议减少细胞移液以减少细胞死亡和轻轻重悬细胞，使得3×10 6个细胞/ ml在流动注射仪。要注意的是更高的细胞浓度会降低细胞流并可能仪堵塞的流动。另一方面，较低的细胞浓度将需要较长的处理时间。由此一可能需要调整取决于TY浓度细胞的PE进行分析。 备选测定法:部署以记录在四个大类别的离子通道的细胞表面质膜落入存在其他方法:1）共焦成像的方法利用荧光团共轭抗体和靶蛋白之间的高亲和力相互作用; 2）蛋白质化学技术搁在由生物素化的试剂的靶蛋白的共价修饰; 3）非稳态噪声分析的电措施;和4）用放射性探针靶蛋白的光亲和标记。在电压-门控钙通道，带氚化（±）光亲和标记的情况下- [3 H]的PN 200-100，二氢吡啶家族的高亲和配体进行了广泛的在20世纪80年代31使用。它仍然是一个高度敏感的检测，但涉及放射性物质31，这已经跟不上潮流的来临以下的操作高度敏感的荧光和发光探针尤其是年轻的研究者。此外，它不可能测量在拮抗剂，以使得表面的表达和功能之间的相关性超越了这种方法的范围的存在离子通道活性。在试验连续的另一端，离子通道的非稳态噪声分析需要高质量的千兆欧密封的膜片钳记录与分钟长的录音稳定的基线。用这种方法可以从图形绘制噪声方差为在任何给定电位32-34测定的平均电流的函数的斜率提取在单个小区开放的离子通道的数量。它显示的标准电方法相同的派头。这是除了需要与光谱分析32-34，而不是细胞生物学家的主食一个很好的熟悉劳动密集型和低通量方法。此外，这种类型的analysi的s标识这可能是比在膜总蛋白的数量不同活性离子通道（在打开状态）的数目。用生物素试剂的细胞表面蛋白的标记是一个相对有效的工具来确定存在于质膜的蛋白质。此方法利用由生物素和进一步的高亲和力和高特异性的非共价到膜不通透性的链霉抗生物素蛋白或分子的生物素结合的膜蛋白的共价修饰。这种方法是定性可靠，但受阻或定期使用免疫印迹法揭示蛋白质定量相关问题的限制。共焦成像和其衍生物广泛用于记录膜蛋白与细胞表面35的共定位。定性质膜隔离仍然可以使用差速离心技术，有或无蔗糖梯度36。如在上述流式细胞仪阿萨Y，它依赖于目标蛋白和荧光团共轭抗体之间的高度特异性的相互作用。特别是全内反射荧光成像，是一种有效的方法，可以在膜表面37,38报告单离子通道的分布和分子的行为。读出的绝对是抢眼，但仍低通量小批量的方式。所有这些试验的有其科学价值。高吞吐量流式细胞术为基础的测定是在读出的重现性和可量化指标方面优异，但需要方便地访问多激光流式细胞仪细胞分拣是在大型机构核心设施的一部分。基于荧光板阅读器更便宜，更容易获得，但信噪比与在相同的实验条件下，相同的构建体获得的测量在很大程度上为更高的荧光背景是显著降低。荧光团共轭的成本tibodies也可以在特别是由于因素来的具有与测试构建体来处理适当的控制实验的数目。同一主题的变化包括改变荧光染料标记的抗体以高性价比而酶的活性可以通过化学发光29,30进行测定辣根过氧化物酶或碱性磷酸酶记者抗体。 结论:双色荧光测定法开发来估计使用的流式细胞仪的培养细胞中表达的重组蛋白的相对细胞表面表达。虽然只有两个荧光参数在当前的试验中使用，流式细胞术是经得起超过30的荧光探针的在单个小区中的同时检测，证明该方法的高度灵活性。这种高通量超灵敏测定可适于调查基因突变22,26或翻译后modificati的冲击组件23，以及学习伴侣的药理学特征为膜蛋白的技术39的表面贩卖。这个协议背后的推动力从需要隔离，以评估基因突变对重组​​细胞离子通道的膜表达的影响引起的。要注意，在模型细胞系突变体通道功能障碍的严重程度并不总与在突变携带者5临床症状的严重程度，部分关联，因为在不同的等位基因的突变的组合可能需要触发心脏猝死40是但重要41。在这种情况下，该测定可以看作是提供在与失功能突变22相关联的单个基因研究的单个或多个突变的快速第一步近似。它是然而似是而非今后设想在cardiomyocy的分化的细胞相同的构建体的病毒介导的表达德血统。总体而言，与其他的成像或发光的技术相比，流式细胞仪分选机处理以均匀细胞群体的悬浮液和报告荧光强度的活细胞。这个过程是即使在温和的条件42，而不需要用多聚甲醛，诱导局部等离子体膜通透性的方法固定完成，。该法是快速，特异，并方便以达的分析，以每工作日几百样品。除了分析，细胞可以最终被流式细胞术分选来评估表达较高或较低的水平相同的膜蛋白的细胞的功能的潜力。

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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54732/54732fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Q5 Site-Directed Mutagenesis Kit</td><td>New England Biolabs</td><td>E0554S</td><td>Can be substitute with QuickChange site-directed mutagenesis Kit (Agilent, #200523).</td></tr><tr><td>Tubes 1.5 ml</td><td>Sarstedt</td><td>72-690-001</td><td></td></tr><tr><td>Tubes 15 ml&amp;nbsp;</td><td>Sarstedt</td><td>&amp;nbsp;62-554-002</td><td></td></tr><tr><td>Disposable graduated Tranfer Pipets&amp;nbsp;</td><td>VWR</td><td>160001-192&amp;nbsp;</td><td></td></tr><tr><td>100 mm culture dish</td><td>Corning</td><td>430167</td><td>For standard culture of HEKT cells.</td></tr><tr><td>35 mm culture dish</td><td>Falcon</td><td>353001</td><td>For standard culture of HEKT cells.</td></tr><tr><td>Serological pipette 1 ml</td><td>Sarstedt</td><td>86.1251.001</td><td></td></tr><tr><td>Serological pipette 5 ml</td><td>Sarstedt</td><td>86.1253.001</td><td></td></tr><tr><td>Serological pipette 10 ml</td><td>Sarstedt</td><td>86.1254.001</td><td></td></tr><tr><td>Serological pipette 25 ml</td><td>Sarstedt</td><td>86.1285.001</td><td></td></tr><tr><td>Dulbecco&#039;s high-glucose medium</td><td>Life Technologies</td><td>12100-046</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated, US origin</td><td>Life Technologies</td><td>16140-071</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Life Technologies</td><td>11668-019</td><td>For liposomal transfection. Can be substituted with calcium phosphate transfection.</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-070</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Trypsin-EDTA (1x) 0.05%, phenol red</td><td>Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>1.5 ml microtubes</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>&amp;nbsp;Phosphate Buffered Saline 1x</td><td>Fisher</td><td>BP661-10</td><td>Can be &quot;home-made&quot;.</td></tr><tr><td>Anti-HA FITC conjugated antibody</td><td>Sigma</td><td>H7411</td><td></td></tr><tr><td>IgG1&amp;minus;FITC Isotype Control antibody</td><td>Sigma</td><td>F6397</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td><td>BD Biosciences</td><td>554714</td><td>Fixation/Permeabilization. Permeabilization/Wash solution, store at 4 &amp;deg;C.</td></tr><tr><td>Hemacytometer</td><td>Fisher</td><td>49105161</td><td></td></tr><tr><td>Trypan Blue</td><td>Fisher</td><td>15250061</td><td>To access cell viability.</td></tr><tr><td>Refrigerated Microcentrifuge, 5430R</td><td>Eppendorf&amp;nbsp;</td><td>A14H172200</td><td></td></tr><tr><td>Forma Steri-Cycle CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher</td><td>370</td><td></td></tr><tr><td>Laboratory Platform Rocker</td><td>Fisher</td><td>545034</td><td></td></tr><tr><td>Water Bath</td><td>VWR</td><td>89032-216</td><td></td></tr><tr><td>BD FACSARIA III&amp;nbsp;</td><td>BD Biosciences</td><td>648282</td><td>Flow cytometer.</td></tr><tr><td>FlowJo Software v10</td><td>FlowJo</td><td>FlowJo v10 Dongle</td><td>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的激发和发射光谱表明，FITC被激发与488纳米的激光88％的效率，但效率下降到0％与561纳米的激光器。的mCherry显示其最大荧光的63.9％，当用561纳米激光，但只有7.6％的488纳米激光（ 图3）激发。激发和发射SPECTRA两种荧光团表现出最佳的采光和最小的重叠与选定不同的激光器和过滤器。流式细胞仪分析可提供从一个大的细胞群大约膜蛋白的相对表达的定量信息只要MFI对于给定荧光染料成正比的Ca Vα2δ1对每个单元中的蛋白的表达，而不是荧光细胞的数目。这涉及到使用超出饱和荧光缀合的抗体的浓度。对于抗HA FITC缀合的抗体结合的Ca Vα2δ1（ 图4）滴定曲线示出了三个阶段。虽然在第一阶段中没有检测到荧光，荧光强度与在第二阶段增加抗体浓度成倍增加。饱和度（第三阶段）的点之后，增加抗体的浓度对相对荧光INTEN没有影响减到的FITC（RFI）。的抗HA FITC缀合的抗体的饱和点在5微克/毫升成立，从而保证在实验过程中的共轭抗体的浓度不限制MFI。 加标签的Ca Vα2δ1was在TSA-201cells表达和流式细胞测定法中的FITC偶联的抗HA的存在下，在非透化的细胞（ 图5）进行。适当的控制（ 表1）上的流式细胞仪上的属性的xy值给每个小区通过激光束进行分析。细胞分布上观察点图（ 图5A）。 FCS / SSC散点图确认细胞样品的制备提供了高存活率（ 图5BA）的单细胞悬浮液。对活细胞（P1）的所选择的栅极代表分析的细胞的总数的75％。总蛋白表达Ø在TSA-201细胞˚F钙Vα2δ1被认为是成比例的mCherry荧光。正如mCherry 与 FITC等高线图和直方图可见-与pmCherry钙Vα2δ1-HA转（ 图5BB BC），47 2％TSA-201细胞被发现显著表达mCherry荧光以及显示由于显著FITC荧光的结合在非透化的细胞的Ca Vα2δ1的外部HA标签。无抗体或用同种型进行阴性对照实验表明，FITC仅或主要结合到正转染的细胞。 该协议被用来表征对Ca Vα2δ1的总和细胞表面的表达中TSA-201cells 23 N-糖基化的作用。实验是在非透化的细胞中进行，以评估细胞表面表达并在透化的细胞以评价总蛋白表达以及确认HA抗原决定基的可及性。基于计算出的Ca Vα2δ1的相对表达于MFI估计每个荧光团（mCherry或FITC）。对于突变体ΔMFI值标准化为测定当天为mCherry钙Vα2δ1-HA WT中的最大值相同的条件下表达。这在抗HA的FITC缀合的抗体的绝对荧光强度随时间占的变化是重要的。如在图6A中看到的，在非透化的细胞的ΔMFI为FITC强劲用于WT和4xNQ构造，但接近为16xNQ构建体的背景荧光水平表明该蛋白质是从质膜几乎不存在。测定细胞透之后进行的，结果表明，16xNQ构建体的总蛋白表达也显著降低它是否在透化的细胞，或从非透测定的构mCherry荧光和透化的细胞（ - D 图6C）从FITC荧光推断。如在图6B中看到的，蜂窝的自发荧光水平细胞透，这防止完好非透和透化的细胞之间的绝对荧光值的比较之后增加。对于mCherry测量的ΔMFI荧光是两种不透和渗透细胞以表明通透的解决方案不会改变mCherry的相对荧光信号相似。的N-糖基化位点的突变与在mCherry的荧光强度支承公布观察该蛋白质生物合成/稳定性降低23下降有关。在这一系列的实验中，在透化的细胞测定FITC的相对荧光信号原来到bË比mCherry表明该蛋白的N-糖基化类型的状态可以影响由在胞外域的FITC偶联的抗HA抗体检测出的荧光强度的相对信号更小。共细胞透前后的变化FITC的相对荧光提供了可靠的指标来量化蛋白表达。 <img alt="图3" src="/files/ftp_upload/54732/54732fig3.jpg" /> 图3:FITC和mCherry形成一对合适的荧光染料为双色流式细胞测定法激励（空曲线）和FITC标记的发射光谱（实心曲线）激发488nm的蓝色激光（AA）和mCherry兴奋561纳米黄光。绿色激光（AB）。FITC和mCherry荧光收集用530/30 nm和一个二十零分之六百一纳米bandpasS表示过滤器。不同激光器（有色的垂直线）和滤波器（彩色矩形）的波长被选择为最佳的光收集和最小重叠。 （B）mCherry-钙Vα2δ1-HA蛋白的示意图。钙Vα2δ1倍加标记来表示，可以通过抗HA FITC标记的抗体和组成的荧光细胞内mCherry荧光检测到细胞外无荧光抗原HA。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54732/54732fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54732/54732fig4.jpg" /> 图4:抗HA FITC缀合的单克隆抗体结合的Ca Vα2δ1滴定 TSA-201细胞中瞬时与pmCherry钙的V＆染<html> ＃945;2δ1-HA和与一系列抗HA FITC缀合或同种型的抗体的孵育。仅FITC信号在该面板被分析。 （A）的单参数直方图显示细胞在y轴的相对数量和在x轴为浓度的抗HA FITC缀合抗体的范围（0.01〜20微克/毫升）的FITC荧光强度。 （B）中的抗HA FITC缀合的抗体作为FITC的MFI的函数的半对数座标图。滴定曲线是使用一个网站绑定方程趋势线值R 2 = 0.99561装。对于同型抗体滴定进行显示无荧光预期。 0.001〜0.01微克/毫升的浓度从背景噪声区别。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54732/54732fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1"&gt; <img alt="图5" src="/files/ftp_upload/54732/54732fig5.jpg" /> 图5:代表流式细胞术pmCherry钙Vα2δ1-HA（A）的流式细胞仪分析样本用于浇注战略示意图转非透TSA-201cells的分析。采集与适当的软件后进行数据分析。第一门控策略利用了前向散射（FSC）和侧向散射（SSC）散点图来显示栅极（P1）围绕活细胞和消除任何碎片，死细胞，或聚集体具有比活细胞不同的前向散射和侧向散射。的mCherry（y轴） 对 FITC（x轴）荧光强度的两参数等高线图中显示为P1的人口。长方形闸被放置在小区的自体荧光的边缘以选择阳性群P2（FITC和mCherry）和阴性群P3。在t荧光水平他在区域内的P2细胞和P3用随后的细胞计数与 FITC或mCherry荧光强度的单参数柱状图进行可视化。在Y轴和X轴的荧光强度的转变分别提供的Ca Vα2δ1的总和膜表达的可靠指标。荧光信号的强度增加作为荧光分子数的函数。 （BA）代表FCS / SSC点图的是规定每个条件。一共有10,000个事件被收购进行分析。椭圆门（P1）是由眼周围排除低FSC（死细胞）或高SSC（聚合）的事件活细胞绘制。 （BB）代表双参数轮廓与 FITC（x轴） 的荧光强度。盖茨的mCherry（y轴）的曲线图被放置在所述细胞偏析FITC-mCherry阳性细胞群的自体荧光的边缘（P2栅极）和负荧光的细胞（P3）。 （BC）单巴rameter相对数（或最大的百分比）细胞与荧光强度（FITC或mCherry）的直方图。为P2栅极（荧光阳性细胞）内的细胞测得的荧光强度的分布示于灰色，和荧光强度的中存在的P3栅极（荧光阴性细胞）的细胞的分布显示为一个叠加透明的灰色情节 。可以看出，荧光强度强为mCherry和FITC表明钙Vα2δ1能很好地表达清楚地存在于细胞膜。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54732/54732fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54732/54732fig6.jpg" /> 图6: 氮 -glycosylation网站同时突变打乱了细胞表面的前钙Vα2δ1的PRESSION，稳定TSA-201钙Vβ3细胞中瞬时染pCMV钙V 1.2 WT和pmCherry钙Vα2δ1-HA WT或突变体转染同时进行。如上所述非透化（A）和透化的细胞（B）中，用抗HA FITC缀合抗体染色。对于抗HA的FITC缀合抗体染色（右图）的荧光强度的分布的mCherry 与 FITC荧光（左小图）和直方图的代表两参数等高线图中示出用于后N个 -glycosylation突变体（NQ）四个位点的破坏（4xNQ:N92Q / N184Q / N468Q / N876Q）和16个网站（16xNQ:N92Q / N136Q / N184Q / N324Q / N348Q / N468Q / N475Q / N585Q / N594Q / N663Q / N769Q / N812Q / N876Q / N883Q / N973Q / N986Q）在完整的非透或NP细胞（A）和透化或P细胞（B）。该DISTRIB为P2栅极（荧光阳性细胞）内的细胞测得的荧光强度的ution示于灰色，和荧光强度的中存在的P3栅极（荧光阴性细胞）的细胞的分布是在一个透明显示为叠加灰色的情节 。如在（B）中看到的，自发荧光水平细胞透后增加。对于FITC指示所有转染HA标记的Ca Vα2δ1蛋白质进行染色并通过抗HA FITC抗体检测出的荧光强度为所有转染的透化的细胞增加测量（视为在x轴右移）。 （C）棒图显示FITC的完整的非透（表面表达）存在测量的标准化MFI或透细胞（共表达）。相对荧光密度一式三份测定各条件（30,000个细胞），并在细胞中的三个不同批次transfec泰德历时1个月，因此该数据被显示为平均值±SEM 90,000细胞为每个条件。峰荧光强度转染后24和36小时之间达成。在完整的非渗透细胞测定FITC的ΔMFI值被用作钙Vα2δ1-HA的细胞表面表达的索引，并在透化的细胞测定FITCΔMFI值反映了总蛋白表达（细胞表面和细胞内蛋白质表达）。 ΔMFI为FITC通过从FITC阳性细胞（P2）的荧光强度减去FITC阴性细胞（P3）的FITC荧光强度计算。用同样的方法来计算mCherry的ΔMFI。 MFI值标准化为测定当天为mCherry钙Vα2δ1-HA WT中的最大值。 （D）棒图显示的归ΔMFImCherry非透或透细胞（TOT测量人的表达）。结果表示为平均值±SEM <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54732/54732fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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 ...

determination-relative-cell-surface-total-expression-recombinant-ion

Nanyang Technological University

Research

JoVE Journal

Biology

13.2K 次观看.  Montreal Heart Institute Research Centre. 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。 

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

Measurement of BK-polyomavirus Non-Coding Control Region Driven Transcriptional Activity Via Flow Cytometry

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals are currently not available, methods measuring the impact of potential antiviral agents are required. Here, a dual fluorescence reporter that allows the analysis of the BKPyV non-coding control-region (NCCR) driven early and late promoter activity was constructed to quantify the impact of potential antiviral drugs on viral gene expression via tdTomato and eGFP expression. In addition, by cloning BKPyV-NCCR amplicons which in this protocol have been exemplarily obtained from the blood-derived DNA of immunocompromised renal transplanted patients, the impact of NCCR-rearrangements on viral gene expression can be determined. Following cloning of the patient derived amplicons, HEK293T cells were transfected with the reporter-plasmids, and treated with potential antiviral agents. Subsequently, cells were subjected to FACS-analysis for measuring mean fluorescence intensities 72 h post transfection. To also test the analysis of drugs that have a potential cell cycle inhibiting effect, only transfected and thus fluorescent cells are used. Since this assay is performed in large T Antigen expressing cells, the impact of early and late expression can be analyzed in a mutually independent manner.

In this manuscript, a protocol is presented to perform FACS-based measurement of BK-polyomavirus transcriptional activity by using HEK293T cells transfected with a bidirectional reporter plasmid expressing tdTomato and eGFP. This method further allows to quantitatively determine the influence of novel compounds on viral transcription.

BK-多瘤病毒非编码控制区域驱动转录活性通过流细胞测定的测量

Polyomaviruses, like the BK-polyomavirus (BKPyV), can cause severe pathologies in immunocompromised patients. However, since highly effective antivirals ...

科研

遗传学

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. 
This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. 
The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A necessary step in anticancer aptamer development is to test its binding to the target. We demonstrate a flow cytometric-based assay to study this binding, emphasizing the importance of including a negative control aptamer and cancer cells that are positive or negative for that particular protein.&#160;

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

癌症研究

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

使用定量流式细胞术分析荧光标记蛋白与人工磷脂微泡的相互作用

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

生物化学

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

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

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) ...

生物学

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

使用流细胞测定测量红细胞补充受体1

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

免疫与感染

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

全谱流式细胞术分析原代小鼠T细胞受体诱导的钙内流

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

用于了解输出机制的流式细胞术实验方案

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

作者聚焦：探索癌症进展中 MicroRNA 加载到细胞外囊泡中的机制 

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

使用流式细胞术追踪miRNA释放到细胞外囊泡中

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...