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 Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT) grant SFRH/BD/87256/2012.

1.离子选择性自引用的微电极的制备<ol><li>离子选择性微电极的制备<ol><li>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。 </li><li> Silanize电极以使内表面疏水，以帮助保持所述亲脂性离子载体鸡尾酒。将微电极在金属架和热O / N在烘箱中在&gt; 100℃下使其干燥。机架是金属板用直径为2毫米的孔钻通的方式的一部分。放置在孔中的电极与250向上小费毫升克姑娘烧杯他们。 </li><li>当天上午，关闭烤箱，虽然戴绝缘手套，小心地取出与到位的电极和烧杯中的金属架。关闭烤箱门留住热量。 </li><li>戴乳胶或丁腈手套，实验室外套和保护眼睛。用塑料巴斯德吸管，放置一滴硅烷化溶液I在每个电极的基部（保持烧杯到位;使用浇注唇的用于吸管访问）。硅烷化溶液通过热板汽化和silanizes电极的内部。使用化学提取通风橱这个阶段。放置架/烧杯/电极放回热炉中几小时，以允许任何残余的硅烷化溶液蒸发。 注：为安全起见，不要打开烤箱回。将表明它不能打开，因为它可能含有对人体有害，易燃蒸气烤箱的标签。 </li><li>冷却后，存放微电极微电极的存储罐INSI解用400g干燥剂的玻璃干燥器。微电极可以因而被存储许多星期。 注：另一种方法硅烷化史密斯等人被描述26。 </li><li>背面填充的微电极，用50〜100微升（约1厘米的长度）含有离子的100mM的溶液的被测定（ 见表1和图1B）。在使用本生灯拉一次性塑料巴斯德吸管热罚款细丝。在冲洗卫生署2 O吸管之后，以防止堵塞。 注意：可替换地，调整回填溶液中的离子浓度，以匹配离子的浓度在外部液27。 </li><li>观察在解剖显微镜下的微电极，以确保没有气泡。 <ol><li>如果存在气泡抽头的​​微电极轻轻用手指钉同时保持电极垂直（尖向下）和/或推气泡S OUT的尖端通过施加背压使用改性的硅酮试管更换针的注射器。 </li></ol></li><li>尖填充微电极用15至20 NL离子特定离子载体鸡尾酒（30-50微米的长度）（ 见表1）。放置一个小墨滴离子载体鸡尾酒在显微镜载玻片上的短边。观察微电极尖端在解剖显微镜下，并将其移向所述显微镜载玻片，直到微电极尖端接触的离子载体鸡尾酒大约只有一半仲丁基。绘制离子载体鸡尾酒到微毛细管压力。 注意：避免离子载体鸡尾酒的一个长列，因为这增加了探头的电阻，可使其容易受到电子干扰（噪音），并且还减慢了响应时间。 </li><li>安装在一条直线微电极保持器的微电极用金1毫米阳连接器和氯化银（Ag +）与电线（ 图1B）。&lt;/ LI&gt; <li>微电极夹固定在头部舞台装在一个三维计算机控制的电子微定位器（ 图1A）。 </li><li>放置在测量溶液适合于将被测量的样品的微电极尖端（生理盐水，培养基， 等等 ），以允许微电极至一个小时或两个，或甚至过夜稳定。 </li></ol></li><li>参考电极的制备<ol><li>参考电极（ 图1C）是相同的毛细管如上。切割用金刚石笔毛细管成长5厘米，长度和火抛光在本生灯火焰两端各1〜2秒。 </li><li>填充这些电极以〜200微升的NaCl的3M溶液，CH 3 CO 2 K（乙酸钾）或KCl，用2％的琼脂糖。选择取决于离子的溶液来进行测量（参考电极不应包含被测量的离子; 见表1）。混合琼脂糖，并将该溶液并加热至在微波几乎沸腾。搅拌溶解琼脂糖（溶液变清）。 </li><li>附加参比电极到塑料巴斯德吸管并绘制热溶液进入毛细管。 </li><li>落电极浸入冷3 M氯化钠，CH 3 CO 2 K或KCl溶液，并存储在此的3M溶液在密封管在使用之前。丢弃气泡任何参考电极。 </li><li>安装在一条直线微电极保持器的参考电极（预填充用3M溶液）与氯化银（Ag +）与丸内和金2毫米阳连接器（ 图1C）与电极和保持器连接到一个手动微定位安装在一个磁性支架上。 </li></ol></li></ol> 2.离子选择性自引用的微电极校准<ol><li>制备含有所关注的离子作为对照品溶液校准解决方案; 见表1 </str翁&gt;。括号即在溶液中的样品将在（ 例如培养基，生理盐水）的浓度。也就是说，一个校准溶液必须含有离子的浓度低于在测量溶液中，和一个更高。 <ol><li>例如，使用盐水含有K + 1毫米。以括号此浓度，溶解氯化钾粉末在去离子水至10，1和0.1mM在连续稀释液的浓度。使用这些校准解决方案。可替代地，使用至少两种这些解决方案。 </li></ol></li><li>浸没离子选择性微电极和在每个校准溶液参考电极，并让电压值记录使用专用软件对应的电压稳定后再进行1至3分钟（ 见表1）。 </li><li>由于软件保存数据（放大器输出）作为一个txt文件，将数据复制到一个电子表格文件。积微电极输出（MV）针对的对数的摩尔离子浓度（ 图2A）。 </li><li>应用线性回归分析，计算能斯特斜率，截距和R 2的值。接受微电极，如果能斯特斜率为58±11毫伏/十年一价离子和29±11毫伏/十年二价离子（阳离子为，能斯特斜率为正，负离子对于为负）。此外，良好的微电极应该有很强的线性相关（R 2&gt; 0.9; 图2B）。 注意：这里使用的放大器的输出毫伏毫伏给人以阅读十倍收益。值必须由十倍进行划分。 </li><li>使用线性回归公式的微电极的原始毫伏输出转换成实际离子浓度（ 图2B）。 </li></ol> 3.验证离子选择性微电极技术<ol><li>制备人造源<ol><li>人工来源毛细管是相同的毛细管作为上面。热拉使用微电极拉出器的毛细如步骤1.1.1。 </li><li>回填这些毛细管用200μl的氯化钠，氯化钾的1M溶液，氯化钙2 2 H 2 O或pH4的缓冲液中。选择取决于离子的人工来源待测溶液（ 见表1）。 注：另外，电极拉大与尖端直径（约20微米）和尖填充相同的解决方案，但含有0.5-1％琼脂糖（琼脂糖将防止任何大流量的解决方案）。 </li><li>装入人工来源毛细管上一个显微和沉浸在用于测量样品中的离子的通量的溶液。留在30分钟的溶液中的人造源到1小时，以允许梯度的稳定化。 </li></ol></li><li>离子选择性微电极的验证<ol><li>从该溶液中的人造源的毛细管用于measu浸没离子选择性微电极约一厘米远重离子对样品的通量和闭合电路与参考电极如前。让电压值使用专用软件进行1到2分钟的记录对应的电压稳定后再进行1至3分钟。这个值对应于缓冲器的值（在文献中也称作参考，背景或空白值）。 </li><li>从人造源移动离子选择性微电极至约5微米，并让电压值稳定保持1〜3分钟记录使用该软件对应的电压为1至2分钟之前。 </li><li>通过将离子选择性微电极10，20，40，80，160，320，640和1280微米远离人工来源毛细管重复上述步骤。 </li><li>提取的数据作为一个txt文件和值复制到电子表格文件。 </li></ol></li><li>计算对应于该毫伏值以同样的方式作为校准值的离子浓度。绘制值。 没有德：如果离子通量存在时，微电极检测到的两个位置（图3B）之间的离子浓度差。如果人工来源包含比溶液测定的物种的更多的离子，浓度应该高接近比远源，验证离子选择性微电极的能力，以正确地检测离子束的方向（在这种情况流出;用于人造片，具有较低的特定离子的浓度比测量介质，它应该是涌入）。 <ol><li>利用扩散的菲克定律计算离子通量：J- = Cμ（直流/ DX）其中c是溶液中的离子浓度（mol cm -3）的，μ是离子迁移率（摩尔厘米N - 1秒-1） ，和直流是随距离DX（厘米）（ 图2C）的浓度差。离子通量数据在皮摩尔厘米-2 S常常表现为-1或纳摩尔厘米-2秒-1。 注意：通过Smith 等26中所述的离子通量的计算的另一种方法，都可以使用。主要差异包括使用的扩散系数，代替的离子迁移和背景离子通量（也电压漂移或校正因子）从离子通量测量在盐溶液中没有样品计算的减法。 </li><li>绘制对从源（ 图2D）的距离的每个步骤的离子通量的平均值。远离源，观察磁通值验证所述离子选择性微电极以感测离子通量的不同大小的能力的指数下降。 </li><li>做人工来源验证一次为每个特定的离子旨在被记录，以验证其正确的方向和大小的测量用大信号到n瓦兹比。 注意：没有样品缓冲器的离子通量测量指示背景电平或噪声。通常情况下，缓冲区的测量显示的离子浓度导致非常小的流量，显示易变的方向没有明确的波动。 </li></ol></li></ol> 4.准备测量室注：在实验中，考虑到待测量的样品，以及如何将样品要被安装并固定于微电极的测量。 <ol><li>对非洲爪蟾卵母细胞测量剪切一个800微米的尼龙网（nitex目）的1厘米的正方形，并把它粘成的塑料培养皿（ 图1E）。 </li></ol> 5.离子通量测量<ol><li>测量离子浓度存在于用于执行上以同样的方式作为用于校准溶液的样品的测量结果的缓冲器。X.蟾卵母细胞需要马克的修改林格氏液（MMR）。 Dissolve氯化钠，氯化钾，氯化钙，氯化镁和HEPES到去离子水中，以达到（毫米）的最终浓度：100氯化钠，2氯化钾，氯化钙2，1氯化镁和5 HEPES。用NaOH调节缓冲液的pH至7.5。 </li><li>放置样品到测量室，并使用微定位以限定微电极（ 图3A）的关闭位置使离子选择性微电极靠近样品（大约10微米的距离）。 </li><li>开始使用专用软件的关闭位置和离开的位置从样品（远）之间的微电极的低频率（0.3赫兹）偏移（100微米）。确保微电极的移动是在垂直于样品的表面上。 注意：微电极的偏移可以在软件设置。大偏移增加读允许在测量时更容易探测小通量梯度延长采样间隔，降低了时间分辨率。见<st荣&gt;图3A的例子。 </li><li>使用该软件开始录制。微电极暂停在每个位置和电势以mV被记录在计算机上。获得的测量为至少2分钟，使信号稳定。对于短的时间推移实验，在整个时间过程感兴趣的位置，记录潜在的变化。 </li><li>提取的数据作为一个txt文件和值复制到电子表格文件。 </li><li>计算对应于该毫伏值以同样的方式作为校准值的离子浓度。绘制值。 注意：如果一个离子通量存在时，微电极检测到的两个位置（ 图3B）之间的离子浓度差。 </li><li>通过计算扩散菲克定律离子通量和以前一样（步骤3.3.1）。 </li><li>测量新样品前重复缓冲器的测量，并重复通量测量和计算的步骤，对每一个新的样本。 </li></ol> 6.统计分析和数据显示<ol><li>测试和/或时间使用混合效果的28 ANCOVA模型的控制条件下离子流的位置的独立影响。 注意：协方差分析（ANCOVA）是通过使将用作独立变量都分类和连续措施混合正规ANOVA和回归的一般线性模型。另外，在诱导每个人和最终嵌套效果重复测量相关误差的存在下，混合效应模型被用来模拟的固定和随机效应准确的估计。 </li><li>计算使用组水平与Bonferroni校正的学生t-检验多个测试28两两比较。 </li><li>箱线图生成根据位置和时间总结离子通量测量。包括描述成对学生 t P值上述（ 图3D），并指示P值的显着性水平如下：*：P &lt;0.05; **：P &lt;0.01; ***：P &lt;0.001 29 </li></ol>

The authors declare that they have no competing financial interests.

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?...

<html>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“&gt;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&gt; 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。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IonAmp&#160;</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>none</td>
			<td>amplifier created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>IonAmp32&#160;</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>none</td>
			<td>software created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Headstage pre-amplifier</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>INA116</td>
			<td>BSR Voltage Follower INA116, designed by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>MicroStep Driver</td>
			<td>&#160;BioCurrents Research Center, Woods Hole, MA, USA</td>
			<td>none</td>
			<td>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 &#8220;XBL function matters&#8221; (http://www.xuyue.org/) or from &#8220;YoungerUSA&#8221; (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Manual micropositioner&#160;&#160;</td>
			<td>World Precision Instruments&#160;</td>
			<td>Model KITE-R</td>
			<td>Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Magnetic stand&#160;&#160;&#160;</td>
			<td>World Precision Instruments</td>
			<td>Model M10</td>
			<td>Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Vibration isolation table&#160;&#160;</td>
			<td>Newport Inc.&#160;&#160;&#160;&#160;&#160;</td>
			<td>Model VW-3036-OPT-023040</td>
			<td>Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/)</td>
		</tr>
		<tr>
			<td>Part of three dimentional micropositioner: angle bracket, 90&#176;, slotted faces</td>
			<td>Newport Inc.&#160;&#160;&#160;&#160;&#160;</td>
			<td>Model 360-90</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Part of three dimentional micropositioner: Peg-Joining Dovetail Stage 0.5 inch X Travel</td>
			<td>Newport Inc.&#160;&#160;&#160;&#160;&#160;</td>
			<td>460PD-X</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Part of three dimentional micropositioner: Quick-Mount Linear Stage, 0.5 inch XY Travel</td>
			<td>Newport Inc.</td>
			<td>460A-XY</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Kwik-Fil thin walled borosilicate glass capillaries without filament&#160;</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW150-4</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Electrode puller&#160;</td>
			<td>Narishige</td>
			<td>&#160;PC-10</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Metal rack</td>
			<td>Made in-house</td>
			<td>none</td>
			<td>Metal electrode holder made in-house by drilling 2 mm wide holes half centimeter spaced in a 10cm by 15cm rectangular base of steel</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>QL</td>
			<td>Model 10 Lab Oven</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Silanization solution I&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>85126</td>
			<td>Hazardous, handle as recommended by provider&#160;</td>
		</tr>
		<tr>
			<td>Glass Petri dish; Pyrex</td>
			<td>Fisher Scientific</td>
			<td>316060</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Electrode/micropipette storage jar</td>
			<td>World Precision Instruments&#160;</td>
			<td>E215</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Glass dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-595E</td>
			<td>Contains Drierite dessicant (W.A. Hammond Drierite Co. Ltd, Xenia, OH, USA). Place petroleum jelly on the seal to make it airtight.</td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette&#160;</td>
			<td>Fisher Scientific</td>
			<td>11597722</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Fisher Scientific</td>
			<td>S97329</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Sigma-Aldrich</td>
			<td>S8902</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Straight microelectrode holder</td>
			<td>Warner Instruments</td>
			<td>QSW-A15P</td>
			<td>with a gold 1 mm male connector and Ag/AgCl wire</td>
		</tr>
		<tr>
			<td>Straight microelectrode holder&#160;</td>
			<td>World Precision Instruments</td>
			<td>MEH3S</td>
			<td>with a AgCl(Ag+)pellet inside and a gold 2 mm male connector&#160;</td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>VWR</td>
			<td>60872-306</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Nitex mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX750</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Glue; Loctite epoxy</td>
			<td>VWR</td>
			<td>500043-451</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Deionized water&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>99053</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Sodium Hydroxyde</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1190</td>
			<td>none</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>none</td>
		</tr>
	</tbody>
</table>

measurement of extracellular ion fluxes using the ion-selective self-referencing microelectrode technique

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。

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?...

Watch this Scientific Journal Video about Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique at JoVE.com

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

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.

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

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

measurement-extracellular-ion-fluxes-using-ion-selective-self

Research

JoVE Journal

Biology

13.9K 次观看.  University of California, Davis. 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.

视频: Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Recapitulation of an Ion Channel IV Curve Using Frequency Components

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|&lt;0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.

There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

重演的离子通道IV曲线的使用频率分量

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

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 ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

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 ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...