研究实验室是由弗里茨 - 本德基金会，慕尼黑，德国（以TD）和威腾/黑尔德克内部研究资助大学（以W.-KL）资助。我们要感谢博士教授弗兰克博士Thévenod（研究所生理学，病理生理学和毒理学，威腾大学/黑尔德克）的有益的建议。

1.准备试剂和解决方案<ol><li>制备改性的汉克平衡盐溶液（HBSS）（以mM计:136.9氯化钠，氯化钾5.37，0.34的Na 2 HPO 4，0.44 KH 2 PO 4，4.17的NaHCO 3，pH为7.2，无酚红）。保存在4℃。 </li><li>作的1.5M的CaCl 2和2M 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）（pH7.2）中使用蒸馏水储备溶液。 </li><li>制备丙磺舒的250mM的储备液。为游离酸形式（通常被称为水不溶性），溶解丙磺舒粉末在1N的NaOH，填充到四分之三，用蒸馏水将终体积的和缓慢滴定至pH 7.4，用1N HCl洗涤。保存在4℃。 </li><li>溶解荧光膜可渗透钙的无水二甲亚砜（DMSO）中结合染料到原料浓度为1mM。储存于-20°C黑暗。 </li><li>之前的每个实验中，新鲜制备ħ含丙磺舒-由BSS的磁通（PHF）缓冲液结合所改性的HBSS以（在终浓度）为1.5mM的CaCl 2，10mM的HEPES，pH值7.2，5％胎牛血清，2mM的丙磺舒，和5.55毫米的葡萄糖。加热至37℃。 </li></ol> 2.细胞培养<ol><li>每一个6孔板或35mm培养皿中的标准培养基中的孔板2.5×10 5个肾近端小管细胞（WKPT-0293 Cl.2）和生长2天达到约70％汇合。 </li></ol> 3.钙染料荷载<ol><li>对于每个孔或皿中，混合含有5％胎牛血清，4μM的细胞可渗透的钙结合染料和2mM丙磺舒1毫升培养基。 </li><li>替换用1ml样染料混合物在各培养基中井和在湿润的培养箱中，用5％的CO 2孵育在37℃下进行30分钟。覆盖板用铝箔，以防止荧光指示剂的漂白。 </li></ol> 4.准备铈LLS的流式细胞仪<ol><li>温暖的0.05％胰蛋白酶 - 乙二胺四乙酸（EDTA）溶液至37℃。 </li><li>从每个孔中吸出钙样染料溶液和加入1 ml胰蛋白酶沿着井的壁。 </li><li>孵育在37℃，直到所有的细胞被分离。 </li><li>转移细胞到1.5ml管中，沉淀经离心分离微量离心以10000×g，1分钟。 </li><li>小心吸出上清液，而不会干扰沉淀。 </li><li>通过加入1ml的PHF缓冲和重悬通过上下吹打洗涤细胞。 </li><li>通过以10000×g离心1分钟沉淀细胞。 </li><li>重复步骤4.6。 4.7。进一步的2倍。 </li><li>重悬细胞于500微升的PHF缓冲液至终体积并在1小时内使用的细胞用于实验。 </li><li>可选:计数单元，以确定细胞浓度，并使用5×10 4 - 1个10 5个细胞每测量。 </li></ol> 5.流动细胞色素ometer设置<ol><li>开始的流式细胞仪中，打开抽屉射流和翻转通气阀拨动开关，以增加鞘液罐的空气压力。 </li><li> "素"的仪器，以从内管和流动室中除去潜在居住气泡。 </li><li>在流式细胞仪的软件，打开两个散点图窗口。 <ol><li>在第一散点图需要向前散射光（FSC）为用于Y参数来控制细胞的样品中，通过它们的大小和它们的粒度，分别质量X参数和侧向散射光（SSC）。 </li><li>第二点图测定钙结合染料的荧光强度。选择用于使用对数标度和时间以秒为单位的线性尺度的X参数在Y参数的合适的荧光检测通道。 </li></ol></li><li>设置流速为&#39;高&#39;。 </li></ol> 6.流式细胞仪Measurement <ol><li>执行该测量在室温下。在流式细胞术样品管（11.5×75mm的），加入480微升的PHF缓冲器。 </li><li>加入20μl细胞悬液，并简要旋涡混合。 </li><li>移动管支撑臂的一侧，并通过样品注射管定位在样品管中，直到一个紧密的密封而形成。返回管支承臂至其原始中心位置。 </li><li>通过调整荧光信道，例如优化测量该样品的平均荧光强度是在约10 2上的y轴。保存并用于随后的实验。 </li><li>要启动一个实验，重复步骤6.1至6.3。 </li><li>记录基线荧光为50秒。 </li><li>暂停测量，取出样品管中，加入的兴趣，涡流化合物简要并返回到样品注入管。这一步不应该超过15秒。 </li><li>恢复测量并继续，总共204.8秒。 </li><li>使用钙我onophore，如伊屋诺霉素（10μM），和一个钙离子螯合剂，如乙二醇四乙酸（EGTA），的MnCl 2或氯化钴（2毫摩尔），对阳性和阴性对照。 </li></ol> 7.数据分析<ol><li>分析使用专用的流式细胞仪软件进行离线分析数据，如WinMDI（Windows多文档界面的流式细胞仪），由乔·特罗特在斯克里普斯研究所，流式细胞仪核心设施开发的免费软件。 </li><li>打开一个散点图与SSC和FSC参数。 </li><li>选择使用"区域"工具的细胞进行分析的区域（在图像上单击鼠标右键），以排除死细胞或细胞碎片从数据分析的左下角找到（ 图1）。 </li><li>象限分析（ 图2） <ol><li>打开随时间的x轴方向和荧光强度在y轴的密度曲线图。使用的所有事件。 </li><li>美国Ë同门区域在7.3。 </li><li>通过使用"象限"工具量化的数据。 </li><li>调整象限隔板，使得垂直线被放置在化合物加成的时间和水平行被放置在所述的控制基线荧光的中心。确保该象限隔板的定位是等于在所有样品中。 </li><li>细胞在每一个象限中的百分比显示在每个角（ 图2）。比较样品之间的右上象限（UR）。 </li></ol></li><li>直方图分析（ 图3） <ol><li>以直方图的窗口与在x轴和事件上，y轴的荧光强度打开控制数据。使用的所有事件。 </li><li>对于图案的概述，被比作对照区的叠加图（转到文件→打开文件→选择文件→叠加）添加测试数据。 </li><li>对于定量分析，单直方图赢DOWS必须使用。门使用区域R1从步骤7.3的细胞群。 </li><li>在未经处理的对照，使用标记功能（"标记"）来标记从最小值和对照峰的最大荧光和结束处的最大荧光强度之间的中点开始的区域进行分析。细胞在这个区域中的百分比来计算。 </li><li>检索统计窗口的数据。重复使用的分析相同标记的区域剩余的数据文件。 </li></ol></li><li>荧光强度分析（ 图4） <ol><li>打开点图以时间上的x轴方向和荧光强度在y轴上。使用的所有事件。 </li><li>启用"动力学模式"和"覆盖动力学线"。蓝线将出现在散点图表示平均荧光强度。 </li><li>通过创建多个矩形区域量化的相对钙离子内流。每个区域应该有AW约"50"，IDþ相当于"10秒"。定义多达15个矩形区域。 </li><li>从统计数据窗口检索量化和复制到电子表格中。 </li><li>用的y平均数据进行分析。计算R 2至R 6，这相当于20〜50秒的平均值。这是基线值。 </li><li>选择的钙离子内流分析（取决于起病，持续时间和强度），适当的时间间隔。 </li><li>计算每个区域中的钙信号的这个时间间隔相对于所述平均基线值的范围内，也就是化合物加成，其被设置为100％之前的荧光信号。确定的平均值和各区域的标准偏差张贴化合物加成;这是通过将化合物诱发的平均钙信号。 </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

钙是在充当第二信使信号传导分子，也可以作为内质网和线粒体之间进行通信的装置，多种细胞过程的关键事件。来测算胞浆游离钙离子浓度的能力是可以应用于细胞生物学的各个领域一个非常有用的技术。存在各种方法来检测细胞内钙信号。经典地，从一个比例钙指示剂，如呋喃-2信号，在使用荧光成像的设置和绝对钙浓度的活细胞监测可衍生自确定的最小和最大荧光强度。但是，这种技术需要专门的设备，如荧光显微镜，活细胞成像设置和分析软件。在这里，我们描述了在使用流式细胞仪粘附上皮细胞的胞质溶胶中，是访问大多数实验室对钙检测的简单方法。 上皮细胞是宝ssession负责的代谢物，异生素和药物输送转运的阵列。特别重要的是SLC22A超家族中，向其中有机阳离子和有机阴离子转运蛋白属于22和ATP结合盒（ATP）的超家族，其计数的多药耐药性的P-糖蛋白ABCB1和多药耐药蛋白（梅纳反应）其成员23之中。使用大鼠肾WKPT-0293近端小管细胞系Cl.2初始实验是通过Gergely的等人的论文中描述进行。16。这些细胞被带入悬挂和装有荧光 - 上午03点。然而，只有适度的变化，观察10微米离子霉素（38.14％和50.95％，在控制由象限分析或1.78倍，多个区域分析）。我们假设，上皮运输负责为穷人装载细胞的荧光 - 上午03点。为了解决这个问题，丙磺舒和PSC833，有机一个抑制剂信联盟转运蛋白和多药耐药P-糖蛋白，分别同时与钙结合染料对细胞单层使用。丙磺舒改进的钙结合染料加载，通过增大基线荧光强度所指示的，而PSC833是没有效果的，这是相对于在中国仓鼠卵巢细胞24以往报告。这种差别可能在于ABCB1在不同细胞系的水平;该WKPT-0293 Cl.2细胞系包藏ABCB1的低的内源性水平，其可以通过的有毒物质，如有毒金属25,26来诱导。使用丙磺舒提高钙的染料负载可以扩展到表现出丙磺舒敏感的运输系统的其它细胞类型，例如神经系统，血 - 脑屏障和肝脏，在钙信令可以在胞内信号传导中发挥作用的细胞途径。 最简单的检测钙结合荧光 - 3在于其requi种类调和只有一个激发波长（506纳米）和一个发射波长（525纳米）。然而，与单波长的钙结合染料，作为一个非比例的染料中产生的数据，只能被用于量化相对增加细胞内钙超过未处理的对照。要确定绝对的钙离子浓度，比例式染料是必要的，因为光谱发生偏移的钙结合后，允许纠正不平等染料加载和漂白。该比例染料印-1已被用于检测的钙动员免疫细胞通过流式细胞仪27,28但到目前为止，这尚未在上皮细胞中进行。使用的紫外线激发的印度支那1中的两个发射光谱（400纳米时的钙结合并当无钙475纳米），绝对细胞内钙浓度所描述的Grynkiewicz 等人 11可以被确定。 我们的例子中的数据采用药物化合物诱导APermanent改变细胞内钙。生理性钙离子信号强度低，显示快速动力学和是暂时的，因此，问题仍然是流式细胞术方法的流程是否足够敏感，可以探测生理性钙信号。实验与ATP进行的WKPT-0293 Cl.2细胞系，其中藏着嘌呤受体主要表现在提出申请的ATP增加钙信号（由活细胞成像测量）。使用流式细胞仪，增加细胞内钙可以被1观察 - 100μM的ATP，但加上恢复测量的延迟之后化合物加成指该信号的仅一部分可以被记录的信号的快速性。因此术方法的流程的一个主要缺点是其有限的容量来记录快速（&lt;10秒）和瞬时钙信号;而该方法更适用于刺激其产生速度较慢的瞬态信号（&gt; 10秒）或持续升高的计算值IUM信号。这些限制可通过减少刺激浓度或通过施加抑制剂的钙再吸收的机制保留在细胞质中释放的钙，但一定要小心，以选择的抑制剂浓度不增加细胞内钙本身被规避。 收购活细胞成像的信息和流式细胞仪有很大的不同。用活细胞成像，高分辨率意味着钙的空间变化可以通过选择单个细胞内的区域进行成像测量。此外，荧光信号不间断获得每1 - 2秒，因此迅速钙信号可以被记录下来。与此相反，流式细胞术是更适合于慢和长效钙信号和从全细胞群检测钙信号。仅在钙信号的相对变化可以定量用流式细胞仪法的流动。但是，流式细胞仪法的流动具有许多ADVAN每日新闻与传统的活细胞成像:1）高通量的化合物（刺激剂和钙信号传导抑制剂筛选）可以执行; 2）具体的成像专长不是必需的; 3）研究的技术人员可以进行测量; 4）不具备该装置的临床和非临床实验室进行高分辨率成像技术和比例钙测量可以调查钙信号。两者合计，流式细胞术方法的流程是有用的，如果研究者最初希望建立对钙的作用，可以很容易地适用的抑制剂，以确定上游和下游信号传导途径，而活细胞成像就需要以表征钙信号（ 例如 ，动力学，强度，空间的变化），更详细。 综上所述，我们描述了一种快速，简便，灵敏的方法对细胞内钙实时相对变化的检测难以负荷附着上皮肾细胞，并已纳入停牌。对于实验者不访问流式细胞术，该方法可以容易地应用到与细胞悬浮液在反应杯一荧光分光光度计。

钙是负责细胞的生理学和病理生理学信号1的发送的重要的第二信使。细胞内钙的浓度保持在较低水平，通过钙泵和钙交换体的活性。肌浆/内质网钙ATP酶（SERCA）重新充质网（ER）钙库作为"泵泄漏"系统而质膜钙ATP酶（PMCA）挤出钙向细胞外隔室的一部分，无论是在ATP依赖的方式2。生理信使，例如激素或神经递质，通过G-蛋白偶联受体，激活磷脂酶C，其水解磷脂酰肌醇4,5-二磷酸（PIP2）在质膜中，以产生二酰基甘油和三磷酸肌醇（IP3）3的发送它们的信号。而二酰基甘油保留在质膜上，IP3扩散进入细胞溶胶和结合的IP3受体秒（IP3Rs），它们的配体激活的钙通道，在ER膜和钙被发现从ER腔商店最终于增加细胞内钙浓度的4释放。对于钙到达胞质溶胶的替代路线是通过钙离子通道和热交换器中存在的质膜。这两个车厢之间的串扰进行了描述:钙诱导钙释放（CICR），其中的细胞外钙离子诱导的ER店5钙释放和存储操作的钙进入（SOCE），其中排空ER店是由STIM感应到，并导致开放Orai的钙通道在细胞膜，促进ER商店6的再填充。 在病理情况下，细胞的钙反应是放松管制和细胞内钙增加的情况下的生理刺激1。钙反应可能会影响在许多方面:长期钙信号，延迟钙去除胞浆钙商店或钙局部变化的枯竭。此外，线粒体承担了中心作用在缓冲和释放钙7。的延长和/或超大的细胞内钙的增加导致细胞死亡。事实上，钙是往往不是参与细胞病理生理反应，是在疾病，如神经变性疾病，心脏疾病，癌症，骨疾病，肾疾病，这主要与细胞死亡有关的各种各样的键事件和丢失或改动的器官或组织功能8-10。此外，钙信号的扰动已与细胞适应和改变的细胞功能和响应。 传统上，钙信号被测量与被加载到细胞中的细胞可渗透的乙酰氧基甲基（AM）酯形式的带负电的荧光钙指示剂。一旦通过细胞酯酶切割上课时，荧光指示器保持在胞内区室中，增加的荧光强度时，钙绑定。最知名的和使用的钙指标是比例的Fura-2罗杰钱永健和他的同事开发出11。用不同的亲和力对钙的钙指标允许不同的钙池进行监控。检测方法包括活细胞成像，荧光酶标仪，流式细胞仪。钙信号的相对快速动力学（通常几秒钟至几分钟内），使活细胞成像获取关于钙信号的特性的最信息的最佳方法。除了 ​​钙信号（在几毫秒内）的快速动力学，活细胞成像是单细胞12内研究钙信号的细胞区室化的较好方法。绝对钙浓度可通过测定钙的ind的最小和最大荧光来计算icator通过添加钙离子螯合剂和透化的细胞，分别如由Grynkiewicz 等人 11。 首先被开发于1980年代在免疫细胞中的使用流式细胞仪来测量钙信号，其中的机会，以测量多个参数，例如膜完整性和细胞群的分离，并且细胞在悬浮液中的要求并结合单波长的发展钙指标方面流式细胞仪的理想和convenientdetection方法13-15。在贴壁细胞，活细胞成像提供钙信号传导，其中最重要的动力学的信息最多，但需要复杂的设置，其包括荧光显微镜，灌注系统，维护蜂窝环境，如温度，并专门显微镜软件，用于获取和分析数据。替代的方法如荧光酶标仪或药理学通过使用钙离子螯合剂ogical手段所无法比拟的获得信息的条款。流式细胞术变得越来越广泛地用于监测在贴壁细胞的细胞内钙虽然在大多数的研究中，测量只进行终点。在免疫的B细胞16使用由Gergely的等最初开发的方法。通过流式细胞仪具有实时动力学和监测从一个样品群体的单个细胞的荧光强度的变化在细胞内游离钙离子浓度已成功地在肿瘤上皮细胞中测得的和癌症干细胞杂种17。该方法还适合于贴壁的上皮细胞，这是难以负荷与钙指标18使用。 这里，使用从大鼠肾近端小管（WKPT-0293 Cl.2）19的S1片段衍生的永生化粘附上皮细胞系，我们描述了一种优化的简化米ethod用于通过流式细胞术改变细胞内钙浓度的测定。因为许多上皮细胞具有许多的阴离子化合物的有机转运，细胞装载钙指示剂的可以被证明是一个挑战。为了防止钙的指标，丙磺舒，有机阴离子转运原型抑制剂和最初开发来降低青霉素20的肾脏排泄外排，是在培养液同时使用。将细胞维持在单层钙指示剂装载，带入悬浮液和钙信号可化合物加成后，立即检测到。

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:79px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:321px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-3, AM</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">F-1241&#160;</td>
			<td>Cell permeable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Probenecid</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8761</td>
			<td>Water insoluble, dissolve in 1 N NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ionomycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thapsigargin</td>
			<td style="width:79px;">Tocris Bioscience</td>
			<td style="width:119px;">1138</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tunicamycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7765</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSCalibur Flow Cytometer + CELLQuest software</td>
			<td style="width:79px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Windows Multiple Document Interface for Flow Cytometry (WinMDI)</td>
			<td style="width:79px;">Joe Trotter, The Scripps Institute</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">WKPT-0293 Cl.2 rat kidney proximal tubule cell line</td>
			<td style="width:79px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Made available by Dr. Ulrich Hopfer (Department of Physiology &#38; Biophysics, Case 
			Western Reserve University, Cleveland, OH)</td>
		</tr>
	</tbody>
</table>

cytosolic calcium measurements in renal epithelial cells by flow cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

以增加细胞内钙，2药理化合物施用于装载细胞渗透性的钙结合染料的Fluo-3-AM肾近端小管细胞（WKPT-0293 Cl.2）。未处理的对照样品中填装的钙结合染料在丙磺舒的存在，并进行混合，但不加任何化合物。毒胡萝卜素（TG）是SERCA泵导致泄漏出的ER和导致增加的胞内钙的经典抑制剂。衣霉素（TUN）的块的N-糖基化的蛋白质，导致激活未折叠蛋白应答的，但也增加了细胞内钙18,21。作为阳性对照，离子霉素（IONO），钙离子载体制备的质膜透对细胞外钙，被使用。为了分析，将细胞群体（ 图1中R 1）选通由SSC / FSC点图中选择的区域。为荧光强度的变化的密度曲线图对比时间为每个数据文件的创建。通过使用象限功能，点图被分离成代表前和化合物加成后与上方和下方的基线荧光的Fluo-3荧光强度四个区域。作为定量门控细胞群体中的每个象限的比例确定允许增加细胞内钙离子浓度。在这个例子中，3μM的TG引起的增加的细胞中的右上象限表示一个钙信号已经被诱导的百分比。细胞的比例从对照42.8％提高到51.3％（1.20倍），TG处理的细胞。类似地，6微米TUN增强细胞具有增加的钙荧光强度的百分比，以49.9％（1.17倍）。导致细胞中的右上象限（ 图2）的65.5％（1.53倍），10μM的IONO应用。直方图分析得到1.41倍，1.36倍和2.11倍，用TG，TUN和IONO，分别为（图3）（用于统计，参见&lt;SUP&gt; 18）。最后，使用多域分析模式，TG TUN和IONO造成1.55倍，1.29倍和4.54倍的增加钙信号切换控制，分别为（ 图4）。 最敏感的分析模式是多个区域的分析。与此相反的象限和直方图的分析模式，其中所述细胞群的分布进行量化，所述多个区域的分析模式量化从总细胞群中荧光信号的变化。在从不同的分析模式所得出的最大差异是对于离子霉素是从1.53倍以上的控制从象限分析，以4.54倍，从所述多个区域的分析范围。测试的化合物没有表现出这样的变化。然而，在所有的分析模式，钙信号的程度诱发保持不变，虽然绝对值不同， 即 IONO有最大的影响其次是TG，然后TUN。 </p&gt; 导致细胞内钙永久性更改TG，TUN或IONO中的应用。以确定是否生理钙信号，这是暂时的，可以使用这种方法来测定，ATP的涂布WKPT-0293 Cl.2细胞。 ATP诱发的快速和短暂的钙信号，这可能不是由流式细胞术方法的流被记录的全部内容。 ATP的浓度为20 - 100微米难以因此，检测浓度降低到"慢下来"的钙信号。 如图5所示，在细胞内钙的变化可以加入5μMATP后进行测定。尽管如此，没有记录荧光变化的峰值。使用象限分析（ 图5B）和直方图分析（ 图5C）的模式，在钙信号中的ATP没有差异可以是由于钙信号的瞬时性质进行检测。然而，随着跟踪的选定部分，所述多个矩形，R区分析模式记录在钙信号中的1.85倍后，立即重新开始测量（ 图5A）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51857/51857fig1highres.jpg" width="500" /> 图1. SSC与FSC点图 。该细胞群的完整性，使用侧向角散射（S​​SC）和前向散射（FSC）进行评估。的区域被选中时（R1），用于进一步分析。死细胞和细胞碎片，从而减少了光的散射，被排除在外。如图代表点图。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51857/51857fig2highres.jpg" width="500" /> 图2.象限分析 。钙结合染料吸附WKPT-0293 Cl.2细胞的基线荧光测定为50秒。测量被停止，化合物或二luent溶液，涡旋混合并测量立即被恢复为总共204.8秒。使用象限分析进行定量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51857/51857fig2highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51857/51857fig3highres.jpg" width="500" /> 图3.直方图分析 。钙结合与TG，TUN或IONO处理染料吸附WKPT-0293 Cl.2细胞的直方图。使用标记功能，门控单元具有较高的钙结合染料的荧光强度进行定量定位标记的起点在控制曲线的峰值和最大荧光结束的百分比。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51857/51857fig3highres.jpg" target="_blank">请点击此处查看LARG</a>呃版本这个数字。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51857/51857fig4highres.jpg" width="500" /> 图4.多区域分析 。钙结合染料吸附WKPT-0293 Cl.2细胞的基线荧光测定为50秒。测量被停止，化合物或稀释剂中的溶液，涡旋混合并测量立即被恢复为总共204.8秒。使用多个矩形区域分析进行定量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51857/51857fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51857/51857fig5highres.jpg" width="500" /> ATP诱导的钙信号的图5.分析 。基线荧光Ø˚F钙结合染料吸附WKPT-0293 Cl.2细胞测定为50秒。测量被停止，为5μMATP的加入，涡旋震荡以混合和计量立即被恢复为总共204.8秒。使用多个矩形区域分析（A）中 ，象限分析（B）或直方图分析（C）的数据进行了量化。为多个区域的分析，只有短暂的钙信号进行分析。代表性的数据或从n个分析= 3 - 7所示<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51857/51857fig5highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

钙是参与许多生理学和病理生理学的信号通路。活细胞成像，需要专门的设备和非常耗时。使用流式细胞仪来确定在纳入悬浮贴壁的上皮细胞内钙的相对变化快速，简单的方法进行了优化。

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

cytosolic-calcium-measurements-renal-epithelial-cells-flow

Nanyang Technological University

Research

JoVE Journal

Biology

15.2K 次观看.  University of Witten/Herdecke. 钙是参与许多生理学和病理生理学的信号通路。活细胞成像，需要专门的设备和非常耗时。使用流式细胞仪来确定在纳入悬浮贴壁的上皮细胞内钙的相对变化快速，简单的方法进行了优化。 

视频: Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

科研

生物学

Flow Cytometry Purification of Mouse Meiotic Cells

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 unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...