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?...

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 border="0" cellpadding="0" cellspacing="0" width="352">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
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			<td height="22" style="height:22px;width:88px;">Papain</td>
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			<td style="width:88px;">39S11614</td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
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			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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		<tr height="22">
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		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
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			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">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

8.7K 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.