这项工作是支持由斯洛文尼亚Z2 - 9229项目和程序P2 - 0249的研究机构。这个视频代表的“电穿孔技术为基础的技术和处理”科学研讨会和研究生课程，组织，斯洛文尼亚卢布尔雅那大学电气工程学院每两年一次的补充材料。

第一部分：预备步骤<ol><li>在本实验中使用中国仓鼠卵巢细胞（CHO - K1）。实验室TEK二钱伯斯（2井，4厘米，每个2）（Nalge NUNC，德国）在0.7x10〜5细胞/毫升辅以8％胎牛血清的HAM - F12培养基，0.15 mg / ml的细胞均镀L -谷氨酰胺，16毫克/毫升的庆大霉素（Sigma - Aldrich公司，Steinheim，德国）和200个单位/毫升Crystacillin（Pliva公司，萨格勒布，克罗地亚），并在5％的CO 2在37 ° C 。此外，细胞也可以镀上玻璃盖单（0.13至0.16毫米厚）涂多聚赖氨酸，如蜂窝胶粘剂，。 </li><li>在其培养基培养的细胞。持续2至4小时产量细胞仍然大致呈球形，而是一个小部分，他们的膜牢固地附着在表面的孵化。另外，16日至20小时的潜伏期后，细胞完全附着在表面，并有更复杂的形状，但其中大部分仍不能分割。 </li><li>准备加入843μL二甲基亚砜（Sigma - Aldrich公司，Steinheim，德国）5毫克原Invitrogen公司小瓶染料，10毫米的DI - 8 - ANEPPS（Invitrogen公司，尤金，俄勒冈州，美国）股票的解决方案。在4 ° C原液可存储数个月，在一台冰箱。在开始实验之前，热身的解决方案，直到二甲基亚砜晶体溶解。 </li><li>有些细胞系可能需要使用的嵌段共聚物，以纾缓细胞膜的染料掺入。聚醚可购买20％DMSO（F - 127，Invitrogen公司，尤金，俄勒冈州，美国）原液，或相同浓度的原液，可以由溶解在DMSO嵌段准备。库存嵌段共聚物的解决方案，可在室温下保存。 </li></ol>第二部分：载入细胞与DI - 8 - ANEPPS <ol><li> 10毫米的DI - 8 - ANEPPS和2.5μL20％在1毫升的微调修改的最低限度的基本中等SMEM（钙耗尽版）（中等M8167或M4767，Sigma - Aldrich公司，德国Steinheim，嵌段共聚物混合3μL在1.5 mL Eppendorf管）。这就产生了“加载的解决方案”，包含约30微米的DI - 8 - ANEPPS和0.05％的嵌段共聚物。对于其他类型的细胞，其原生文化传媒可用于代替SMEM。 </li><li>替换为加载的解决方案在实验室- Tek腔室的培养基。传输室冰箱10分钟，在4 ° C。在此温度下，染料通过质膜的内化，在很大程度上抑制。 </li><li>染色后，轻轻洗净多余的染料纯SMEM两到三倍。 </li><li>保留在会议厅内的1.5毫升的SMEM。 </li></ol>第三部分：实验和图像采集<ol><li> （在我们的例子中，德国蔡司ZEISS AXIOVERT 200）与油浸物镜（x63，NA 1.4），单色（多彩四，Visitron，德国）和一个冷却CCD相机（VisiCam配荧光显微镜观察细胞1280，Visitron，德国）。 MetaFluor 7.1.1图像采集和处理MetaMorph 7.1.1（两个分子器件，Downingtown，PA，USA），但也可用于其他类似的数据采集软件。激发波长在490 nm处的采集软件设置，并选择一个适合ANEPPS排放过滤器，例如，一个带通滤波器，中心在605纳米（605/55m，分色565 DCXR，色度，罗金厄姆，佛蒙特州，美国）。如果单色不可用，在490 nm为中心的激发滤光片，可以用来代替。 </li><li>放置在显微镜舞台上的细胞室，在箱体底部位置的电极，将它们连接到脉冲发生器。介质覆盖的电极。 </li><li>为了保持细胞活力，减少加热，脉冲应该足够低的幅度和持续时间短。在本实验中使用的直流电源电压和一个定制的微处理器控制的切换装置与35 V幅度和持续时间50毫秒（足够低，以避免电）的方波产生。另外，商业的脉冲发生器，如33210A（安捷伦，美国加利福尼亚州圣克拉拉），连接到放大器或商业的刺激，如S88（草，西华威，扶轮社，美国），都可以使用。脉冲传递到两个并行的Pt / Ir电极丝，它们之间的0.8毫米直径4毫米的距离，创造一个电场的V /约88厘米之间的电极和诱导ΔΦ。在本实验中所用的电极之间的确切场分布描述[1]。 </li><li>找到感兴趣的细胞。套用单一的电脉冲或脉冲序列。每个脉冲，获取荧光图像：一个紧接脉冲（控制图像），并在脉冲（脉冲形象）。由于DI - 8 - ANEPPS的低反应，在荧光的变化是很难通过肉眼辨别，成为明显的ONLy在处理。必须同步脉冲传递图像采集，这是一个采集软件的功能。为了避免漂白和可能加热，光照可以限制脉冲持续时间。 </li></ol>第四部分：图像处理和分析<ol><li> MetaMorph软件中打开图像。每个脉冲，减，在控制和脉冲图像的背景。 </li><li>选择一个单元格，设置地区的利益，所以它对应的膜。沿着这个区域中的控制和脉冲图像测量的荧光强度，并转移到电子表格中的值。 </li><li>每个脉冲，脉冲数据减去控制数据，并除以控制数据结果来获得相对荧光的变化。如果一个脉冲序列，确定每个脉冲的相对荧光变化值可平均获得更可靠的测量。 </li><li>变换成ΔΦ相对使用荧光校准曲线的变化。一个粗略估计，这条曲线可以从文献中获得，但更高的精度，它必须为每个特定的安装测量。对于本实验所用的细胞和设置在荧光下降6％，相当于100ΔΦ[2] mV的增加。 </li><li>最后，情节的电压，或如Excel（微软公司雷德蒙，华盛顿州，美国），西格玛积（SYSTAT软件公司，美国加利福尼亚州圣何塞）在图形软件包的功能相对弧长原产地（OriginLab公司，北安普顿，马萨诸塞州，美国）。也可以使用一个合适的过滤器，如移动平均滤波器，平滑的曲线。 </li></ol>

<ol>
	<li>Maz&#232;res, S., &#352;el, D., Golzio, M., Pucihar, G., Tamzali, Y., Miklav&#269;i&#269;, D., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non+invasive+contact+electrodes+for+in+vivo+localized+cutaneous+electropulsation+and+associated+drug+and+nucleic+acid+delivery.">Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery.</a> J. Control. Release. 134, 125-131 (2009).</li><li>Pucihar, G., Kotnik, T., Vali&#269;, B., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+determination+of+transmembrane+voltage+induced+on+irregularly+shaped+cells.">Numerical determination of transmembrane voltage induced on irregularly shaped cells.</a> Annals Biomed. Eng. 34, 642-652 (2006).</li><li>Huang, C. J., Harootunian, A., Maher, M. P., Quan, C., Raj, C. D., McCormack, K., Numann, R., Negulescu, P. A., Gonzalez, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+voltage-gated+sodium-channel+blockers+by+electrical+stimulation+and+fluorescence+detection+of+membrane+potential.">Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.</a> Nat. Biotechnol. 24, 439-446 (2006).</li><li>Bedlack, R. S., Wei, M., Fox, S. H., Gross, E., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+electric+potentials+in+soma+and+neurite+membranes.">Distinct electric potentials in soma and neurite membranes.</a> Neuron. 13, 1187-1193 (1994).</li><li>Cheng, D. K. L., Tung, L., Sobie, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonuniform+responses+of+transmembrane+potential+during+electric+field+stimulation+of+single+cardiac+cells.">Nonuniform responses of transmembrane potential during electric field stimulation of single cardiac cells.</a> Am. J. Physiol. 277, H351-H362 (1999).</li><li>Teissie, J., Eynard, N., Gabriel, B., Rols, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+cell+membranes.">Electropermeabilization of cell membranes.</a> Adv. Drug. Del. Rev. 35, 3-19 (1999).</li><li>Sersa, G., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemotherapy+of+tumours.">Electrochemotherapy of tumours.</a> JoVE. 22, (2008).</li><li>Kotnik, T., Bobanovi&#x0107;, F., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+transmembrane+voltage+induced+by+applied+electric+fields+a+theoretical+analysis.">Sensitivity of transmembrane voltage induced by applied electric fields a theoretical analysis.</a> Bioelectrochem. Bioenerg. 43, 285-291 (1997).</li><li>Schwan, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+tissue+and+cell+suspensions.">Electrical properties of tissue and cell suspensions.</a> Adv. Biol. Med. Phys. 5, 147-209 (1957).</li><li>Gowrishankar, T. R., Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+electrical+modeling+of+single+and+multiple+cells.">An approach to electrical modeling of single and multiple cells.</a> Proc. Natl. Acad. Sci. USA. 100, 3203-3208 (2003).</li><li>Joshi, R. P., Hu, Q., Schoenbach, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+studies+of+cell+response+to+ultrashort,+high-intensity+electric+fields+-+Implications+for+intracellular+manipulation.">Modeling studies of cell response to ultrashort, high-intensity electric fields - Implications for intracellular manipulation.</a> IEEE Trans. Plasma Sci. 32, 1677-1686 (2004).</li><li>Gross, D., Loew, L. M., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+membrane+potential+changes+induced+by+applied+electric+fields.">Optical imaging of cell membrane potential changes induced by applied electric fields.</a> Biophys. J. 50, 339-348 (1986).</li><li>Fluhler, E., Burnham, V. G., Loew, L. M. S. p. e. c. t. r. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=membrane+binding,+and+potentiometric+responses+of+new+charge+shift+probes.">membrane binding, and potentiometric responses of new charge shift probes.</a> Biochemistry. 24, 5749-5755 (1985).</li><li>Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dyes:+measurement+of+membrane+potentials+induced+by+DC+and+AC+electric+fields.">Voltage-sensitive dyes: measurement of membrane potentials induced by DC and AC electric fields.</a> Bioelectromagnetics Suppl. 1, 179-189 (1992).</li><li>Hibino, M., Shigemori, M., Itoh, H., Nagayama, K., Kinosita, K. . J. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+conductance+of+an+electroporated+cell+analyzed+by+submicrosecond+imaging+of+transmembrane+potential.">Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.</a> Biophys. J. 59, 209-220 (1991).</li><li>Lojewska, Z., Franks, D. L., Ehrenberg, B., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+effect+of+medium+and+membrane+conductance+on+the+amplitude+and+kinetics+of+membrane+potentials+induced+by+externally+applied+electric+fields.">Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.</a> Biophys. J. 56, 121-128 (1989).</li><li>Zhang, J., Davidson, R. M., Wei, M. D., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electric+properties+by+combined+patch+clamp+and+fluorescence+ratio+imaging+in+single+neurons.">Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.</a> Biophys. J. 74, 48-53 (1998).</li><li>Montana, V., Farkas, D. L., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+ratiometric+fluorescence+measurements+of+membrane-potential.">Dual-wavelength ratiometric fluorescence measurements of membrane-potential.</a> Biochemistry. 28, 4536-4539 (1989).</li><li>Knisley, S. B., Justice, R. K., Kong, W., Johnson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometry+of+transmembrane+voltage-sensitive+fluorescent+dye+emission+in+hearts.">Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts.</a> Am. J. Physiol. Heart Circ. Physiol. 279, H1421-H1433 (2000).</li><li>Pucihar, G., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-dependent+numerical+model+of+transmembrane+voltage+inducement+and+electroporation+of+irregularly+shaped+cells.">A time-dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells.</a> IEEE T. Biomed. Eng. 56, 1491-1501 (2009).</li></ol>

在不同的实验设置，如膜电压门控通道，动作电位的传播，刺激心肌细胞或细胞膜电[3，4，5，6，研究诱导膜（跨膜）电压，ΔΦ，测量也很重要， ，7]。通过简单的细胞形状，ΔΦ可以计算分析。例如，对于一个球形细胞，ΔΦ是由施万方程，哪些国家的电压是成正比的磁场强度和细胞大小和余弦函数如下沿膜[8，9]。对于更复杂的细胞形状，ΔΦ可以偏离很大的余弦，必须确定或者数字，用计算机[2，10，11]，或实验，使用一个电位染料[12，13，14，15]。 广泛用于这一目的的电位染料之一是DI - 8 - ANEPPS（DI - 8 -丁基 - 氨基 - 萘乙烯吡啶 - 丙基磺酸钠），依赖于膜电压的激发和发射光谱的快速染料，这使细胞膜上，来衡量其价值的ΔΦ变化的非侵入性观察。在这段视频中，我们将展示使用DI - 8 - ANEPPSΔΦ测定的实验方法。 染料由教授莱斯利勒夫和他的同事[13，14]在康涅狄格大学，属于快速反应染料类。 DI - 8 - ANEPPS nonfluorescent在水中，并成为强烈的荧光，当它结合到细胞膜的脂质双层。 ΔΦ结果在一个分子内的电荷分布和染料的荧光光谱分布和强度相应的变化的变化的变化。 DI - 8 - ANEPPS的荧光强度的变化成比例的改变ΔΦ;染料的响应线性电压，范围从-280 mV的到+250 MV [4，16]。荧光染料，膜染色不均，相对较小的变化和染料内化的DI - 8 - ANEPPS合适的膜电压，如休息组件的绝对测量，虽然这种努力也报道[17]。然而，它适用于在膜电压变化较大，如发病nonexcitable细胞暴露在外部电场引起的膜电压[12，13]，或在可兴奋细胞[4，5]的动作电位，测量。尽管这里不适用，DI - 8 - ANEPPS还允许ΔΦ决心比例测量荧光激发[18]或排放[19]，从而提高响应灵敏度，降低上述的影响。由于DI - 8 - ANEPPS渍膜，​​它也可以使用显然是作为一种膜标记[2]。 染料的弊端之一是，它是容易漂白，因此，应避免强光的长时间曝光。 （I）钾离子载体缬氨霉素和一组不同的钾浓度在外部介质进行校准染料[2,18]，或（ii）膜片钳电压钳模式[17]。 最后，ΔΦ球形细胞和更复杂的形状细胞的测量，视频演示ΔΦ的幅度和空间分布上的细胞形态的影响。因此，对于球形细胞ΔΦ接近余弦，在与施万的方程协议，而更复杂的细胞形态的空间ΔΦ分布是更复杂的[20] ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>di-8-ANEPPS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3167</td>
<td>potentiometric fluorescent dye</td>
</tr>
<tr>
<td>pluronic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>potassium ionophore</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>SMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M8167 or M4767</td>
<td>Spinner modification of the Minimum Essential Medium</td>
</tr>
<tr>
<td>Ham-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N4888</td>
<td>culture medium</td>
</tr>
<tr>
<td>fetal calf serum</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>&nbsp;</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Lab-Tek II</td>
<td>&nbsp;</td>
<td>Nalge Nunc</td>
<td>155379</td>
<td>chamber</td>
</tr>
<tr>
<td>DC voltage supply</td>
<td>&nbsp;</td>
<td>Elektro-Automatik</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microprocessor-controlled switcher</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>electrodes</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

measuring the induced membrane voltage with di-8-anepps

到外部电场细胞的位置会导致本地的电荷再分配内和在细胞膜附近的细胞外，导致电压穿过细胞膜。这个电压，称为诱导的膜电压（也诱导的跨膜电压，或诱导的跨膜电位差）和ΔΦ表示，只存在只要外部磁场存在。如果休息电压是目前对膜，在其上的感应电压叠加（添加）。通过电位荧光染料，如DI - 8 - ANEPPS之一，它是可以观察细胞膜上的变化ΔΦ和非侵入性来衡量其价值。 DI - 8 - ANEPPS变得强烈荧光，当绑定到用的荧光强度成比例的改变ΔΦ的变化，细胞膜的脂质双层。该视频显示测量的协议ΔΦ使用DI - 8 - ANEPPS，还演示了细胞形态上的幅度和空间分布ΔΦ的影响。

Watch this Scientific Journal Video about Measuring the Induced Membrane Voltage with Di-8-ANEPPS at JoVE.com

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

外加电场诱导细胞膜上的电压，称为诱导膜电压（ΔΦ）。通过电位染料DI - 8 - ANEPPS，这是可能的非侵入性来衡量的ΔΦ。该视频显示测量的协议ΔΦ使用DI - 8 - ANEPPS。

诱导膜电压测量与DI - 8 - ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

measuring-the-induced-membrane-voltage-with-di-8-anepps

Research

JoVE Journal

Biology

17.3K 次观看.  University of Ljubljana. 外加电场诱导细胞膜上的电压，称为诱导膜电压（ΔΦ）。通过电位染料DI - 8 - ANEPPS，这是可能的非侵入性来衡量的ΔΦ。该视频显示测量的协议ΔΦ使用DI - 8 - ANEPPS。

视频: Measuring the Induced Membrane Voltage with Di-8-ANEPPS

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

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

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. ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Methods Article

诱导膜电压测量与DI - 8 - ANEPPS