Name: cytosolic calcium measurements in renal epithelial cells by flow cytometry
Uploaded: 2014-10-28T13:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

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>

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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?...

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><tbody><tr><td>Fluo-3, AM</td><td>Invitrogen</td><td>F-1241&amp;nbsp;</td><td>Cell permeable</td></tr><tr><td>Probenecid</td><td>Sigma-Aldrich</td><td>P-8761</td><td>Water insoluble, dissolve in 1 N NaOH</td></tr><tr><td>0.05% Trypsin-EDTA (1X)</td><td>Invitrogen</td><td>25300-062</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I9657</td><td></td></tr><tr><td>Thapsigargin</td><td>Tocris Bioscience</td><td>1138</td><td></td></tr><tr><td>Tunicamycin</td><td>Sigma-Aldrich</td><td>T7765</td><td></td></tr><tr><td>FACSCalibur Flow Cytometer + CELLQuest software</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Windows Multiple Document Interface for Flow Cytometry (WinMDI)</td><td>Joe Trotter, The Scripps Institute</td><td></td><td>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><td>WKPT-0293 Cl.2 rat kidney proximal tubule cell line</td><td></td><td></td><td>Made available by Dr. Ulrich Hopfer (Department of Physiology &amp;amp; Biophysics, Case&lt;br/&gt; 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，?...

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

Universidad de Cartagena UDEC

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

发育生物学

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

生物学

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 ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

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 ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

监测ER / SR钙释放与目标Ca<sup&gt; 2+</sup&gt;传感器捕捉器<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

<meta charset="utf8"></head><body>用于通道活化研究的单细胞钙成像

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

<meta charset="utf8"></head><body>用于研究钙离子通道激活的单细胞钙成像

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

用于研究钙离子通道激活的单细胞钙成像

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...