细胞外离子流的使用离子选择性自引用的微电极技术测量
1Department of Dermatology, Institute for Regenerative Cures,University of California, Davis, 2Departamento de Biologia, Centro de Biologia Molecular e Ambiental,Universidade do Minho, 3Department of Neurology and Center for Neuroscience,University of California, Davis Imaging of Dementia and Aging Laboratory, 4Department of Ophthalmology, Institute for Regenerative Cures,University of California, Davis
Summary
Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.
Abstract
从动物，植物和单细胞细胞通过称为细胞膜分隔来自外部的细胞质屏障包围。细胞层诸如上皮细胞也形成了从外部或多细胞生物体的不同区室分离的内部的屏障。这些障碍的关键特征是跨细胞膜或细胞层离子的微分分布。两个属性允许这种分配：1）膜和上皮显示选择透过性，以特定的离子; 2）离子通过跨细胞膜和细胞层的泵输送。这些特性在维持组织生理发挥关键作用，并作为信令线索受损后，修理过程中，或病理条件下。离子选择性自引用的微电极使离子的具体助熔剂，如钙，在单细胞和组织水平的钾或钠的测量。微电极包含离子载体鸡尾酒是选择性地透过特定离子。内部填充液含有感兴趣的离子的浓度设定。微电极的电势是由离子的外部浓度来确定。作为离子浓度变化时，微电极的电位变化作为离子活度的对数的函数。当来回移动靠近离子源或宿（ 即在一个浓度梯度由于离子通量）的微电极电位变动在振幅成正比的离子通量/梯度。该放大器放大的微电极信号和输出被记录在计算机中。离子通量然后可以通过使用电极电位变动，微电极的偏移和其他参数扩散的Fick定律进行计算，如特定的离子迁移率。在本文中，我们详细介绍的方法使用离子选择性自引用的微电极来测量细胞外离子流D存在一些具有代表性的结果。
Introduction
所有动物细胞通过脂质双层膜，其分离从外部环境细胞质包围。细胞保持电膜电位，负内，通过离子1主动运输。膜电位是该信元可以利用在膜2进行操作的各种分子器件一个储存能量源。神经元和其他可兴奋细胞有很大的膜电位。钠通道的快速开口折叠膜电位（去极化），并产生被沿着神经元2的长度输送的动作电位。除了这些快速电的改变，许多组织和器官的生成和维护显著长期电势。例如，皮肤和角膜上皮产生和由离子向泵浦（主要是钠和氯）3保持反式-上皮电位和细胞外电流。
帐篷“>虽然内源性细胞外电流的测量使用振动探头4-6，并使用微电极系统7-10允许细胞膜和上皮细胞层的电参数的测量，膜或反式上皮电位测量时，它们不提供任何指示所涉及的离子物种。微电极与选择性离子载体可以测量在溶液特定离子浓度。离子梯度或焊剂可能在不同位置的两个或多个电极来测量。然而，每个探针的固有电压漂移将会不同，从而导致测量不准确，甚至检测的梯度，这是不存在的。在“自参考”模式中使用的单电极，由此它移动在低频率的两个点之间来解决这个问题。现在离子通量可以看出针对相对缓慢和稳定的信号漂移的背景（参见图3B）。 </P>
离子敏感测量系统使用离子选择性自引用的微电极来检测离子接近的组织或单一细胞的小细胞外通量。该系统由一个放大器，其处理从微电极和微步进电机和驱动器的信号，以控制所述微电极的运动。离子选择性微电极和参比电极是闭合电路经由探头前置放大器（ 图1A）连接到放大器。计算机软件确定的微电极的运动（频率，距离）的参数，并记录该放大器的输出。步进电机控制通过一个三维微定位器的微电极的运动。低频振动离子选择性微最早是在1990年到具体衡量的钙流11。以及钙，商业访问离子载体鸡尾酒现已作出MICRoelectrodes钠，氯化物，钾，氢，镁，硝酸，铵，氟化锂，或汞敏感。
基本上，自引用离子选择性微电极技术转换溶解在溶液中成电势的特定离子，这可以通过一个电压表来测量的活性。离子载体鸡尾酒是一种不混溶的液体（有机，亲油）相，用离子交换性能。离子载体选择性配合物（绑定）特定离子可逆地与包含在微电极（电解质）水溶液中和水溶液中的微电极浸渍（ 图1D），它们之间的传输。此离子转移导致电化学平衡和微电极和参比电极之间的电位的变化由电压表测量。根据能斯特E中的电压是正比于特定离子的活度的对数quation允许离子浓度（ 图2A和B）的计算。
目前，几个系统允许离子通量测量使用类似的概念或原理。例如，扫描离子选择性电极技术（SIET）12,13或微电极离子通量估计（米非司酮）技术由Newman和Shabala 14-16开发有市售，以确定特定的离子广泛用于研究界通量在细胞膜和组织在各种动物，植物和单个活细胞模型的发生。离子选择性微电极已经用于测量氢，钾和钙通量穿过植物根部17，氯化物磁通在大鼠脑动脉18和花粉管19， 氢通量在滑冰视网膜细胞20，在小鼠骨21，各种离子的钙通量在真菌菌丝22和r中通量在过程中单细胞角膜伤口23，终于钙流出愈合12,24。另请参见有关离子选择性自引用的微电极25的详细信息如下评论。
下面的文章详细描述了如何准备和执行使用在单细胞水平离子选择性自引用的微电极技术的内源性细胞外离子流的测量。
Protocol
1.离子选择性自引用的微电极的制备离子选择性微电极的制备热拉薄壁的硼硅毛细管，而不使用微电极拉出器长丝（1.5外径，1.12内径）。 注：此给出提示3-4微米的直径。较小的尖端具有较高的电阻，这使得微电极更容易受到电子噪声，并且也与对离子浓度的变化中以较慢的响应相关联。有用的信息可以在由Smith 等人 26出版的论文中找到。 Silanize电极以使?…
Representative Results
钙内流后出现单细胞打伤24我们以前曾表明。因此，我们要求其他离子流是否在单细胞发生伤人。我们使用X.蟾卵母细胞，一个行之有效的模式，单细胞伤口愈合和30-34电生理记录24,35-39。有趣的是，钾离子多集中内部X.蟾卵母细胞（约110毫米）40比中使用的细胞外溶液（以MMR 1×1毫米），表明钾的时受伤外排。为了证实该假说，我们X的过程中测?…
Discussion
用于体内细胞外离子通量测量成功的最重要的步骤是：减少了噪音，离子选择的微电极和参比电极的正确加工，并将样品和两电极的定位。
为了尽量减少噪音，在记录系统应该有接地（接地）法拉第笼优选与金属平顶（隔振）表，该表也接地。此外，该显微镜底盘还应当接地。电噪声的来源包括光源。光纤“风扇的”光源造成最小的电噪声。最后，保持银线和球团在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Science Foundation grant MCB-0951199, and in part by the NIH grant EY01910, California Institute of Regenerative Medicine grants RB1-01417 and by the Fundação para a Ciência e Tecnologia (FCT) grant SFRH/BD/87256/2012.
Materials
| IonAmp | BioCurrents Research Center, Woods Hole, MA, USA | none | amplifier created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
| IonAmp32 | BioCurrents Research Center, Woods Hole, MA, USA | none | software created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
| Headstage pre-amplifier | BioCurrents Research Center, Woods Hole, MA, USA | INA116 | BSR Voltage Follower INA116, designed by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
| MicroStep Driver | BioCurrents Research Center, Woods Hole, MA, USA | none | three MicroStep drivers are required for X, Y and Z-positioning; created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
| Manual micropositioner | World Precision Instruments | Model KITE-R | Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/) |
| Magnetic stand | World Precision Instruments | Model M10 | Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/) |
| Vibration isolation table | Newport Inc. | Model VW-3036-OPT-023040 | Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/) |
| Part of three dimentional micropositioner: angle bracket, 90°, slotted faces | Newport Inc. | Model 360-90 | Assemblage of the three dimantionnal micropositionner requires also Three electric rotary motors for X, Y, Z control, MPH-1 mounting arm with MCA-2 adjustable-angle post and Various Newport connectors and screws to bolt onto vibration table |
| Part of three dimentional micropositioner: Peg-Joining Dovetail Stage 0.5 inch X Travel | Newport Inc. | 460PD-X | none |
| Part of three dimentional micropositioner: Quick-Mount Linear Stage, 0.5 inch XY Travel | Newport Inc. | 460A-XY | none |
| Kwik-Fil thin walled borosilicate glass capillaries without filament | World Precision Instruments | TW150-4 | none |
| Electrode puller | Narishige | PC-10 | none |
| Metal rack | Made in-house | none | Metal electrode holder made in-house by drilling 2 mm wide holes half centimeter spaced in a 10cm by 15cm rectangular base of steel |
| Oven | QL | Model 10 Lab Oven | none |
| Silanization solution I | Sigma-Aldrich | 85126 | Hazardous, handle as recommended by provider |
| Glass Petri dish; Pyrex | Fisher Scientific | 316060 | none |
| Electrode/micropipette storage jar | World Precision Instruments | E215 | none |
| Glass dessicator | Fisher Scientific | 08-595E | Contains Drierite dessicant (W.A. Hammond Drierite Co. Ltd, Xenia, OH, USA). Place petroleum jelly on the seal to make it airtight. |
| Plastic Pasteur pipette | Fisher Scientific | 11597722 | none |
| Bunsen burner | Fisher Scientific | S97329 | none |
| Microscope slide | Sigma-Aldrich | S8902 | none |
| Straight microelectrode holder | Warner Instruments | QSW-A15P | with a gold 1 mm male connector and Ag/AgCl wire |
| Straight microelectrode holder | World Precision Instruments | MEH3S | with a AgCl(Ag+)pellet inside and a gold 2 mm male connector |
| 6 cm Petri dish | VWR | 60872-306 | none |
| Nitex mesh | Dynamic Aqua-Supply Ltd. | NTX750 | none |
| Glue; Loctite epoxy | VWR | 500043-451 | Mix glue and hardener in equal parts in a plastic weighing boat and mix thoroughly. Sets quickly but leave at RT for 24 h for full curing |
| Deionized water | Sigma-Aldrich | 99053 | none |
| Sodium Chloride | Sigma-Aldrich | S7653 | none |
| Potassium Chloride | Sigma-Aldrich | P9333 | none |
| Calcium Chloride | Sigma-Aldrich | C1016 | none |
| Magnesium Chloride | Sigma-Aldrich | M8266 | none |
| Hepes | Sigma-Aldrich | H3375 | none |
| Sodium Hydroxyde | Sigma-Aldrich | S8045 | none |
| Potassium Acetate | Sigma-Aldrich | P1190 | none |
| Agarose | Sigma-Aldrich | A9539 | none |
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Citer Cet Article
Luxardi, G., Reid, B., Ferreira, F., Maillard, P., Zhao, M. Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique. J. Vis. Exp. (99), e52782, doi:10.3791/52782 (2015).