Name: methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell
Uploaded: 2014-10-22T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

This work is supported by a National Science Foundation award (1145581) to LWG.

注意:整个过程是根据美国国立卫生研究院的指导方针进行的，经批准由伊利诺伊大学芝加哥分校的动物护理和使用委员会。 1，解决方案和文化传媒准备<ol><li>保留所有的解决方案在-20°C至六个月。保持培养基在4℃下进行长达3个月。 </li><li>通过混合1毫升ITSX（胰岛素 - 转硒补充）的青霉素 - 链霉素溶液和1毫升到100毫升的DMEM制备100ml培养基。 </li><li>通过混合250毫升DMEM中，加入2.5ml的100mM的CaCl 2，和2.5毫升的50mM EDTA制备的酶溶液。 </li><li>通过混合225毫升DMEM，25毫升胎牛血清，625毫克的白蛋白，和625毫克的胰蛋白酶抑制剂的制备失活的解决方案。 </li><li>准备了154毫米的NaCl，5.6 mM的氯化钾，5.0毫米肝素钠，3.6毫米的碳酸氢钠，5.6毫米的葡萄糖洛克的解决方案。将pH调节至7.3，用NaOH。 </li><li>制备50毫克/毫升的聚-D-赖氨酸（PDL）溶液。用1毫克/毫升的最终浓度大衣盖玻片。 </li><li>制备140 mM氯化钠，5mM的氯化钾，2mM的氯化钙 ，1mM的MgCl 2的，10毫米的HEPES-NaOH和10mM葡萄糖的细胞外溶液。将pH调节至7.3，用NaOH为〜310纳摩尔/ kg的渗透压。 </li><li>准备了50毫米的NaCl，100毫米TEACl，5 mM的氯化钾，2毫米氯化钙2，1毫米氯化镁2，和10毫米的HEPES吸管的解决方案。调节pH值至7.3，用NaOH与〜290纳摩尔/ kg的渗透压。 </li></ol> 2，肾上腺隔离<ol><li>高压釜，对大型和小型清扫剪刀和两对镊子。在整个过程中戴上手套，然后将解剖托盘上一个冰桶。 </li><li>在使用0-3天取小鼠。安乐死的动物用加压的二氧化碳罐后斩首。 </li><li>利用小剪刀来剪下来的小狗的背下从宫颈癌放疗脊椎<html> CAL尾鳍地区。可以肯定表面的皮肤下的切割和未刺穿进入体腔。 </li><li>其次，剥离皮肤远离脊柱，使肌肉暴露在有脊柱的一个明确的说法。从这里出发，让在颈椎脊髓transectional切割，然后进行沿脊柱两侧的两个平行的切口。 </li><li>与一对保持动物的主体钳子，使用其他钳子抓住脊柱和剥离背部脊柱直到肾脏被暴露。 </li><li>此时，可视化对肾脏顶部的肾上腺。然后小心地从每个动物的两个腺体成锥形套管内填充洛克的解决方案。 注:有时腺体会坚持镊子。如果是这样的话，轻轻用另一对镊子在去除从钳子和进洛克溶液的腺体中提供帮助。腺体可保持在该状态下长达2小时。 </li></ol>E"&gt; 3。组织消化<ol><li>在此期间，气泡，用5％CO 2 +95％O 2的酶溶液15分钟，在使用之前。确保平衡的溶液变成从一个明亮的粉红色到粉红色/橙色。 </li><li>木瓜蛋白酶加入到新鲜鼓泡酶溶液在20〜25单位/ ml的最终浓度。在用木瓜蛋白酶的酶溶液孵育每对接头的每个动物为40-60分钟，在37℃。 </li><li>加失活溶液的75％至每个管并在37℃进行另外10分钟。这个孵育灭活木瓜蛋白酶的酶活性。 </li><li>在温育10min，灭活液后，轻轻腺体转移到新标管，然后清洗腺体3X文化传媒。 </li></ol> 4，细胞分离和电镀<ol><li>洗涤后，放置两个腺体成200μL培养基，然后轻轻地滴定腺体和上下贯通的移液管尖与200μl的移液管，直到不再有一大块组织在该溶液中。 注意:当进行滴定，维持腺体在管与来自200μl的移液管尖端向下的力底部和接合组织轻缓地。务必使用缓慢，持续的招，绝不吹打快速或突然。 </li><li>增加一个额外的250微升培养基，使培养液的总体积为450微升。 </li><li>板60微升此含细胞溶液到每七个12毫米PDL-预涂覆的盖玻片，它被放置在一个60×15毫米培养皿中。 </li><li>一次镀，将盘插入，并设定为37℃，并保持5％的CO 2流的稳定/供应30分钟，以确保细胞存活的环境中孵化。孵育后，轻轻地加入5 ml预先温热的培养基放入培养皿中，并返回盘放回培养箱中放置24小时前，细胞公文包ð录音。 注意:通常情况下，可以使用的细胞电生理记录长达4天。 </li></ol> 5，细胞识别和千兆欧密封形成<ol><li>将12毫米盖玻片进入细胞外液。由于整个肾上腺用于细胞制剂，拌入培养与皮质细胞（小和比铬细胞）的调光器的嗜铬细胞（通常是非常明亮的带褐/米色着色和近乎完美的圆形（ 图1）。 </li><li>拉使用可编程的P-97牵引补丁移液器四个阶段。浸枪头到融化的蜡，这将有助于降低玻璃的电容再火波兰尖端"井喷走"这起蜡枪头内。 注意:当装满补丁吸管的解决方案，补丁移液器通常有〜2MΩ电阻。 </li><li>在数米接近补丁吸管靠近电池icrons。实现了小区连接的配置，补丁吸管更靠近的小区的方法中查找任何轻微的细胞变形。轻轻推吸，直到GΩ的密封来实现的。 </li></ol> 6，细胞贴附电容录音<ol><li>一旦GΩ的印章则透露，通过使用EPC-7加膜片钳放大器使细胞贴附记录和两相模拟锁相放大器（见设备附表）。申请为50毫伏（有效值）的振幅和20千赫频率的锁定放大器的正弦波。 </li><li>在1毫秒的时间常数和24分贝设置的锁定放大器的输出滤波器。设置锁定放大器，使得由温和抽吸脉冲产生的瞬态电容变化只在贴片电容（Im）的痕迹，没有投影到补丁电导（Re）中的跟踪显示（ 图2）的相位。 注:请参阅详细的系统重新校准ference 19。 </li><li>识别典型的电容变化由"向下"的步骤（ 图3）。 注:固体录音将被视为一个"阶梯"式的相似与许多向下的步骤，这表明内在单囊泡。打补丁的过程作为一个机械刺激，因为动作电流通常可以从大多数细胞记录。 </li><li>名的文件作为单独的文件夹，包括日期:该文件中/日/年，每个细胞贴附记录作为自己的个人编号（XX）。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

细胞贴附电容测量要求，以顺利取得录音提供高品质的几个关键步骤:1）肾上腺编制可行的，健康的细胞; 2）客运专线涂层的盖玻片; 3）千兆欧密封的形成; 4）该系统的噪声电平;和5）的相位校正。 动物手术，修饰可以潜在地使是调整手术方法，以最适合的灵巧和防止剥离时发生损坏。此外，关于定位，除去肾上腺足够的做法是必要的，这将被用于电生理记录的细胞全部来自?...

突触传递是由含有神经递质的突触小泡的胞吐作用介导的，而这些囊泡必须经过神经末梢内本地吞再循环以维持长期的神经元沟通。由于突触传递在大脑中的重要作用，了解构成突触小泡循环是实现在整个蜂窝通信更好的理解的重要基础的分子机器。在细胞模型系统，肾上腺嗜铬细胞提供了一些最明确的洞察分子机制基础突触囊泡循环。胞吐作用，在神经递质释放的最后步骤，已经极大地研究并通过使用肾上腺嗜铬细胞1,2的检查。事实上，大多数的分子玩家编排的形成，目标，对接和融合分泌颗粒已经确定，由于应用程序股利ERSE技术，嗜铬细胞1。此外，通过提供一个机会以允许参与胞吐作用的蛋白质机械的单泡的分辨率，嗜铬细胞仍然是一个功能强大的模型，以解决囊泡融合3的问题。 细胞贴附电容测量首先在胞吐3中解决单囊泡融合利用。以通过细胞膜导纳测量与小区连接的配置4-7膜片钳技术来检测囊泡一样小〜中直径为60nm已证明的胞吐作用。导纳的定义是多么容易的电路或设备将允许电流流动的措施;它是阻抗的倒数。因此，导纳测量提供的膜电容的理解。这是由小泡膜进入细胞膜的结合来实​​现;这表明掺入改变表面面积8。每个囊泡融合导致膜电容9,10逐步增加。此外，该导纳测量提供了膜电导和胞吐事件3中 ​​的融合孔导。由于这种技术提供了在胞吐过程中识别单囊泡动力学的独特工具，我们的实验室最近应用这个概念，检测单囊泡11,12的内吞作用。 我们特别感兴趣是网格蛋白介导的内吞作用（CME），它已被认为是一个基本的看家成分在许多细胞13和作为突触小泡胞吞作用在神经端子14,15的主通路。 CME是已知的生物学上重要的，但是，它的动力学仍不能很好地理解，由于在监测单数内吞事件的技术限制。考虑到嗜铬细胞和神经元之间的1胞吐机制的相似性，这是PLausible在嗜铬细胞分裂机制可能会适用于突触小泡内吞作用的神经元。细胞贴附电容测量已用于监测个体的内吞事件并分析裂变动力学，其中大多数方法都未能解决。在我们的细胞附着的录音，在20kHz的正弦波叠加在保持电势，并且输出电流是从两相分离成薄膜电导的一个信道和膜电容，在其它信道锁定放大器16 18。从变化的膜电导和电容，可以计算裂变孔隙，这可能对应于管状薄膜颈部的内化泡连接到前囊夹断质膜的动力学。总的来说，这项技术使我们有机会研究囊泡分裂的调控机制CME的过程中。

<table><tbody><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P0899</td><td></td></tr><tr><td>DMEM</td><td></td><td>15066024</td><td>Keep out of UV</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Cover Glass</td><td>Carolina Biological</td><td>633029</td><td>12 mm</td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140122</td><td>100 ml</td></tr><tr><td>Insulin-Trans-Sel-X</td><td>Life Technologies</td><td>51500056</td><td>Only thaw on ICE!</td></tr><tr><td>Papain</td><td>Worthington</td><td>39S11614</td><td></td></tr><tr><td>EPC-7 plus patch amplifier</td><td>HEKA</td><td></td><td></td></tr><tr><td>BNC-2090 data acquisition board</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Igor data acquisition software</td><td>Wavemetrics</td><td></td><td></td></tr><tr><td>P-97 pipette puller</td><td>Sutter Instruments</td><td></td><td></td></tr><tr><td>Microforge</td><td>Scientific Instruments</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Sutter Instruments</td><td>B150-110-10</td><td>Outer diameter: 1.5 mm&lt;br /&gt;
Inner diameter: 1.10 mm&lt;br/&gt;
Length: 10 cm</td></tr></tbody></table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

细胞存活率和千兆欧姆密封的质量是确定细胞贴附电容记录的质量是至关重要的。因此，为促使有效和高效的细胞培养物之前，电生理记录，和典型的活细胞被示出在图1中是至关重要的。实践和时间将是在实现千兆欧姆密封高品质很有帮助。如果人们可以清楚地看到，当在协议5.3步中描述的补丁吸管的临近细胞的细胞变形，有获得高品质的密封更好的机会。 图3显示膜电...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.8K 次观看.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

视频: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

鼠标肾上腺嗜铬细胞分离

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

科研

生物学

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

神经科学

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

小鼠肾上腺的分离、固定和免疫荧光成像

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

免疫与感染

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

渗透胁迫对分泌性囊泡和胞吐作用的监测作用

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

共聚焦显微镜测量肾上腺嗜铬细胞融合孔隙动力学的三种模式

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

原代培养小鼠肠嗜铬细胞的全细胞电生理

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes.
Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte&#8217;s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

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