这项工作是由美国国立卫生赠款2R01 GM53132和P50 GM071558研究院的支持。

1。载入细胞与钙指标<ol><li>中板35mm玻璃底的MatTek菜细胞（或任何其他的玻璃底部观看商会）。 </li><li>如果使用前用10μg/ml的层粘连蛋白大衣原发性心肌商会1天。板心肌细胞（成年犬，新生鼠，或其他生物隔离）35 KB缓冲区（K -逆转台氏缓冲mmol / L的HEPES，140 mmol / L的氯化钾，8 mmol / L的KHCO 3，2毫摩尔/ L的MgCl 2的 KH 2 PO 4，pH值7.5 0.4毫摩尔/ L）和逐步改变M199培养基辅以15％小牛血清和1％streptamycin，0.5％庆大霉素，在37℃孵育° C在5％ 二氧化碳 。 </li><li>孵育细胞与钙绿色上午（1-5微米;分子探针）或任何所需的钙在室温为30-45分钟。盖用铝箔盘，以避免染料的漂白。 </li><li>清洗细胞Leibovitz15 14MM EGTA中等（三次如果要执行低钙实验）或任何适当的观看媒体。 </li><li> Leibovitz15中等containing14mM EGTA孵育30-45分钟，。 </li><li>如果测试抑制剂的影响（即10μMU73122 PLC抑制剂）在第二个孵育。 </li></ol> 2。显微注射细胞<ol><li>培养心肌细胞在玻璃底部MatTek菜（或其他观看商会）为24小时。 </li><li>变化中苯酚 - 莱博维茨- 15 14毫米EGTA（心肌细胞在无钙介质注入，其他细胞可以在培养基中含有钙注射）。 </li><li>准备显微注射的解决方案-我们的研究中使用的一种肽，消除Gαq -小窝相互作用。请记住，只有少量的体积注入细胞（FL），所以必须使用合理集中的股票。我们用DAPI使用2微米Cav3支架，这种肽，钙培养基。 DAPI是蓝光Ë染料污渍的核心，并允许确定已显微注射细胞。许多钙指标的荧光的DAPI不会干扰。量的DAPI是可变的，我们一般使用足够的可视化的核（0.5和2μm） </li><li>准备控制解决方案，其中包含的DAPI - 单独的染料示踪。 </li><li> microinjections使用自拉或市售的针头。实践与染料注射，以确定适当的针参数。我们注意到，不同的细胞可能需要不同的针的直径和显微注射压力。 </li><li>使用自动显微注射系统，例如InjectMan NI2 FemtoJet泵从Eppendorf公司。设置注射压力至90 P I百帕，并赔偿压力 P C至45百帕。将注射时间 t至0.7秒如需要调整注入的参数。 </li><li>执行倒置显微镜（例如蔡司Axiove microinjectionsRT 200M）配备长工作距离相衬的目标（例如空气40倍，第2阶段目标）。 </li><li>注入细胞在快速的方式尽可能多的。检查使用相衬，选择可行的细胞注射的细胞。 </li></ol> 3。联合免疫的研究<ol><li>用PBS洗涤细胞两次，并修复与3.7％多聚甲醛30分钟。如果需要，细胞可以刺激前固定。 </li><li>洗固定细胞，PBS洗3次，0.2％NP40 PBS孵育5分钟。 </li><li>座使用PBS包含1小时4％山羊血清（或其他适当的血清）。 </li><li> 1％的PBS山羊1小时血清，孵育适当稀释的初级抗体的细胞。 </li><li>洗涤细胞三次，除了荧光标记的第二抗体，如Alexa的 - 氟488抗兔或Alexa的氟647抗鼠二抗稀释为150 mM氯化钠，25毫米的Tris，pH值7.6的10分钟到1:1000 1％山羊血清的PBS（请注意，两个不同的蛋白质探测时抗体，必须提出在不同物种和次要必须对相应的物种）。 </li><li>在37 ° C为1小时，用TBS洗三次。 </li><li>查看TBS缓冲液，或另一种合适的观看媒介中的细胞。 </li></ol> 4。快速钙成像<ol><li>使用激光扫描共聚焦显微镜（例如Fluoview 1000奥林巴斯）。 </li><li>使用高数值孔径的目标。 </li><li>设置采集参数。对于钙的绿色使用488 nm激发，通长515排放过滤器。 </li><li>调整像素的时间 - 快速钙测量其设置为2微秒/像素。 </li><li>设置图像的缩放，达到0.05像素尺寸 - 0.3微米。 </li><li>选择的选择在一个特定的地区的细胞。 </li><li>在时间控制器"窗口中选择采集时间为至少1500扫描（以获得所需的时间框架响应 - 1.3毫秒/线将产生2秒）。 </li><li>记录背景阅读之前的刺激。 </li><li>刺激卡巴5微米的细胞，并立即开始记录数据。保持细胞在1毫升Leibovitz15中等，准备在Leibovitz15卡巴和10μM，轻轻地放入盘中添加1毫升</li></ol> 5。数据分析<ol><li>录制的图像的每个像素中提取的强度。保存为一个使用Fluoview 1000软件的表的强度数据。 </li><li>导入到Sigmaplot强度数据或类似的数据分析程序和斌到〜9-40箱的数据。比较对照实验和电子噪音。 </li></ol> 6。代表性的成果: 我们细胞成像的激光扫描共聚焦显微镜LSM510（蔡司耶拿，德国）配备多线激光激发和一个40x/1.2无复消色差透镜水浸泡的目的。在图。 1（ 红色 ），我们的distribution的小窝- 3的抗体，这是重大的结构性caveolar蛋白在这些细胞中9已固定和染色的成年犬心室心肌的小窝。的Caveolin - 3是immunolabelled与Alexa的647和633纳米线从他兴奋:氦氖激光。图像记录使用LP650 nm发射的过滤器。小窝- 3的分布比较Gαq与Alexa的- 488（ 绿色 ）immunolabeled 。 Alexa的- 488 488nm氩离子激光线很兴奋和图像记录BP505 - 530nm发射滤光片。在这些共定位测量，两个荧光基团被激发，在连续使用多轨收购的方式。此过程，最大限度地减少通道串扰。共定位分析的数据是使用蔡司LSM510 - META提供的AIM软件。可以看出，这两种蛋白质共同定位。 要确定如何小窝- 3和Gα之间的耦合<sub&gt; Q影响钙的信号，我们进行了倒置显微镜（IX70，奥林巴斯）加上一个40X的目标（UPlanApo，奥林巴斯，数值孔径共聚焦激光扫描显微镜系统（Fluoview，奥林巴斯，日本东京）钙成像研究1.3），虽然这些测量也可以对Zeis系统进行如上所述。对于这些测试，细胞加载与Ca 2 +绿。我们兴奋与488nm氩离子激光器，染料和收集的图像通过一个LP515排放过滤器。图像帧，收集了500毫秒的间隔。每个像素的强度值分别提取使用奥林巴斯的软件和ImageJ。行扫描模式用于定量分析更快的Ca 2 +瞬变和Ca 2 +火花。在本研究中使用的像素大小为0.1-0.3微米。行扫描率是约数，每行μS百强。在图。 2我们展示了一个CA 2线扫描+绿装心肌其中像素ð以及时间为2μs，该行的时间为142微秒。该图像是由1200线。 扫描的每一行对应的强度波动的 Ca 2 +绿组成的71分，超过142μs范围采取，可以作为钙反应迅速读出。例如，在图。 3中，我们显示的Ca 2 +活动的个人心肌单行，如测得的Ca 2 +绿色荧光强度在几秒钟的时间功能前 （上）和后（下）5微米卡巴从而提高另外细胞内Ca 2 +通过GαQ -PLCβ活动。当多条线路的平均值，卡巴刺激产生的Ca 2〜1.8倍+绿强度。由于实验条件的变化（室温下，激光功率，探测器响应，染料分布，等），这种增加可能不会被视为个别行扫描。强度值（Y轴值），即使是相似的，很显然，在底部的卡巴刺激的情节，更广泛，相应的更持续的钙离子水平，比刺激前看到狭窄的波动峰，峰。这种强度的扩大和相对上升掩盖小，慢振荡。我们的目标是，以更好地区别这些明显不同类型的钙波动。 这些快速的钙波动的另一个例子是如图。 4。顶部的图形显示刺激心室心肌细胞（红色），平均3线的痕迹，显示为黑色的原始行数据。要确定这些钙波动Gαq介导的程度- caveoline3相互作用，我们注射一种多肽不稳定小窝- 3 - Gαq相互作用（即Cav3肽），从而消除了较长的Ca 2 +电流和这些数据显示为蓝色（我们注意到，我们减去Cav3肽强度，使两条轨迹，可以更好地看到500）。 我们进口的Excel文件到Sigmaplot（Jandel公司）的原始数据的列，并进行了直方图分析。在这种分析中，事件的数量分为箱的数目相等（箱）生产直方图。斌的宽度相等的时间间隔。强度插入绑定到不同的时间，使一个特别斌的高度代表的次数，强度发生在特定的时间窗口。由于这种类型的分析只取决于在同一扫描速度收集数据，许多痕迹，可同时进行分析。此外，这种分析是不敏感的染料水平的差异，激光功率等，以及电子和背景噪音的时间尺度发生速度非常快，仅在第1-2箱。 相应的histograMS图的原始数据。 4显示在页面的底部。为控制细胞的直方图分布在一个大量的红线（线是指导眼睛和没有模型是假设）描绘了广阔的时间分布，相应的箱。相比之下，GαQ - Cav3互动肽扰乱细胞的反应箱局限于一个狭窄的滨分布，表明肽的存在消除了持续时间较长通量。微量注射控制肽，不破坏GαQ - Cav3产生一个类似联合国注射细胞 8直方图。 在图。 5，我们比较快速强度波动的Ca 2 +绿（ 左 ）和成年犬心室心肌新鲜，未刺激大鼠乳鼠心肌细胞缺乏小窝（ 右 ）。在这里，分级的强度超过3分钟的时间内相应的直方图<html>在35查看较慢的​​钙事件。注意，乳鼠心肌细胞上显示一个平面显示慢的Ca 2 +活动缺乏的基线，而一个更有条理的基准是成年细胞的快速波动。这些差异是最容易看到的直方图。 <img alt="图1" src="/files/ftp_upload/3505/3505fig1.jpg" /> 成年犬心室心肌Gαq的分布图1。图像与Alexa的- 488 Alexa的- 488 488nm氩离子激光线和图像兴奋immunolabeled录得使用BP505 - 530nm发射滤光片。右上角的是相同的图像，查看immunolabeled小窝- 3 - 647与Alexa的本地化，兴奋与633纳米线从一个他:氦氖激光和记录使用LP650 nm发射滤波器。共定位是橙色的是合并后的图像在扩大图像的权利左下角。 <html> "pdflinebreak"&gt; <img alt="图2" src="/files/ftp_upload/3505/3505fig2.jpg" /> 图2与Ca 2 +绿色生活犬成人心室心肌细胞钙成像加载。共聚焦激光扫描显微镜系统与40X使用488nm氩离子激光器和LP515排放过滤器的目标图像。 1200线组成的图像，像素停留时间为2微秒，像素尺寸为0.1微米。 <img alt="图3" src="/files/ftp_upload/3505/3505fig3.jpg" /> 图3:从加载与 Ca 2 +绿和成像所述一个生活的成年犬心室心肌，和底部的同一单元格除了卡巴5微米的刺激跟踪的强度波动。 <html> / 3505fig4.jpg"/&gt; 图4。顶部线痕迹的一个例子，从生活犬成年心室心肌加载与Ca 2 +绿（红）超过180毫秒，平均3痕迹显示为一条黑线，和一个显微注射的细胞相比，一种肽，扰乱GαQ -小窝3协会（红色）平均三个痕迹是黑色。我们注意到，我们减去400强度计数，以便查看数据。较低的面板相应的直方图，文中所述的分级数据和BIN号是任意的bin计数（或简单地计算）对应的强度，特别斌总额。我们注意到，红色和蓝色线在控制（左）和肽（右）直方图绘制的线条，让便于识别和数据进行比较，并没有假设模型。 files/ftp_upload/3505/3505fig5.jpg"/&gt; 图5。强度波动的Ca 2 +绿色成年犬心室心肌（ 左 ）和新鲜，未刺激的大鼠乳鼠心肌细胞缺乏小窝（ 右 ）和其相应的直方图如下所示。

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没有利益冲突的声明。

我们已经开发出一种方法来查看和隔离生活心肌的快速钙信号。虽然这些测量的实验条件下，曾经被应用到其他细胞系统10以及心肌细胞 11，使用直方图和斌分析可以从很多的Ca 2 +事件被收购的数据。更快的事件，可以很容易地隔离较慢的适应，了解他们的底层机制的模型。培养心肌细胞，许多小学的细胞株，可就难了。虽然人们普遍认为，测量应尽快采取收购后...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> ABCD1234 </td><td></td></tr></tbody></table>

measuring fast calcium fluxes in cardiomyocytes

心肌细胞有多个时间长短不一的Ca 2 +通量，共同努力，优化功能1,2。钙的变化外代理的活动主要是受这是刺激剂， 如乙酰胆碱3,4质膜磷脂酶Cβ-Gαq本地化的途径。最近，我们发现，质膜蛋白质称为小窝5,6域可以坑害激活GαQ 7。这包封的稳定Gαq激活状态，并在长期的Ca 2 +信号在心肌细胞和其它类型的细胞 8作用。我们发现这个令人吃惊的结果，通过规模快速测量动态生活心肌钙反应。简言之，装有一个荧光的Ca 2 +指示剂细胞。在我们的研究中，我们使用的Ca 2 +绿色（Invitrogen公司，在C.）展品钙离子结合后的荧光信号强度的增加。荧光强度，然后记录使用激光扫描共聚焦显微镜扫描模式。此方法允许沿选定的行的像素荧光强度的时间当然的快速采集，生产数百微秒的时间尺度上的时间痕迹。这些痕迹非常快转移到Excel，然后将进行分析Sigmaplot相比，电子噪音，免费染料和其他控制获得的痕迹。解剖的Ca 2 +不同的通量率的反应，我们进行了直方图分析，随着时间的推移，分级像素强度。分级让我们超过500扫描的痕迹和可视化编译的结果在一个单一的情节空间和时间。因此，缓慢的Ca 2 +波难以辨别时，扫描覆盖由于不同的峰的位置和噪声，可不难看出，在分级直方图。在时间尺度的测量速度非常快通量表明在很短的时间，而较长的Ca 2箱的强度分布窄 +波显示分级数据与广泛分布在较长的时间箱。这些不同的时间分布，让我们来剖析在细胞内的Ca 2 +通量的时机，并确定其影响各种细胞活动。

Watch this Scientific Journal Video about Measuring Fast Calcium Fluxes in Cardiomyocytes at JoVE.com

Measuring Fast Calcium Fluxes in Cardiomyocytes

我们提出了一个方法来隔离在生活细胞利用激光扫描共聚焦显微镜慢通量快速（微秒）钙事件。方法措施，由几百个像素单元格中的记录行扫描荧光强度的钙指标的波动。直方图分析，让我们来隔离不同的钙通量的时间尺度。

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

measuring-fast-calcium-fluxes-in-cardiomyocytes

Research

JoVE Journal

Biology

15.1K 次观看.  Queensborough Community College. 我们提出了一个方法来隔离在生活细胞利用激光扫描共聚焦显微镜慢通量快速（微秒）钙事件。方法措施，由几百个像素单元格中的记录行扫描荧光强度的钙指标的波动。直方图分析，让我们来隔离不同的钙通量的时间尺度。

视频: Measuring Fast Calcium Fluxes in Cardiomyocytes

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...