The authors would like to acknowledge the Painless Research Foundation for support of the work. This work was also supported by the NIH grants R01 NS080921-01 and R21 NS079897-01A1.

伦理声明:维护和使用实验动物的所有程序符合UCSF委员会对动物研究的法规，按照美国国立卫生研究院的规定动物使用和保养（出版85的指导方针进行了 - 23日，1996年修订）。加州大学旧金山分校的机构动物护理和使用委员会批准在这项研究中所使用的协议。 1.仪器，解决方案和准备餐具<ol><li>准备人工脑脊液（学联）。 <ol><li>制备500毫升10×低阳离子溶液，和500毫升的10倍的碳酸氢盐溶液（ 见表1和2的溶液制剂）。保存在4℃，使用一个月内。 </li><li>通过将60毫升的10倍低的阳离子溶液用60ml碳酸氢钠溶液制备600毫升新鲜脑脊液中，然后添加480毫升去离子水以达到600毫升最终体积。泡泡学联与卡波（5％O 2和95％的CO 2），用于在使用前至少10分钟。 </li></ol></li><li>从玻璃毛细管拉补丁吸管。 <ol><li>将薄壁毛细玻璃成吸管拉马准备录音移液器。 注:在我们的实验室中，拉马以下设置参数来达到理想的吸管状:热（510），速度（32）。为完整DRG记录了理想吸管形状应具有低的锥角逐渐细长锥形的，这最好通过试验和错误来达到。 </li><li>确保该吸管的电阻为3-5兆欧时填充有内部溶液（见溶液制备表3）。请参考轴突指南测量吸液管电阻18的详细方法。 </li></ol></li><li> 1毫克胶原酶添加到400μl的脑脊液的，得到的13单位/ ml的最终浓度。然后填写与约20微升胶原溶液各玻璃吸管。商店在-20℃下冷冻填充移液管和三周内使用它们。 </li><li>制得200毫升冷学联（约4℃）的。为了冷却学联迅速将其放入烧杯中，用密封在-20℃下冷冻保鲜膜和地点约20分钟，直到它开始冻结。放置在冰浴中和气泡与卡波金的烧杯10分钟。泡在室温其余400毫升学联与卡波。 </li><li>而脑脊液被冷却，切一次性塑料移液管的端部以放大的开口到一个直径为5毫米（这将被用于传输DRG）。收集以下手术器械:手术刀（＃15），梅奥直，弯剪，精2毫米尖咬骨钳，斜角肌挤压（齿）镊子，虹膜剪，弹簧剪刀和两个细镊子。倾含氧冷脑脊液成3玻璃培养皿（外径:10厘米）。 </li></ol> 2. DRG解剖<ol><li>麻醉大鼠（200-220 g）在PE钠ntobarbital（100毫克/千克，IP）并确认麻醉水平是通过测试脚蹬反射和眨眼反射足够。 </li><li>验证将大鼠麻醉后，刮胡子腰部区域和切开沿中线的皮肤和皮下组织。从分离的L1-S2级棘突用细骨膜椎旁肌。 </li><li>用弯剪从L1棘突削减到S2。使用直梅奥剪刀尽在暴露脊柱约为L1和S1横向切口，取出脊柱的这一部分整块并迅速淹没到冷学联（步骤1.5准备），2-3分钟。在排除腰病种付费的，安乐死是通过双边开胸进行，而老鼠仍然是深戊巴比妥麻醉下。 </li><li>冲洗掉从去除脊柱的血液和连接的组织，并转移到陪替氏培养皿用冷脑脊液。 ü瑟细咬骨钳去除薄层。切割沿中线硬膜与microscissors以暴露脊髓。轻轻抬起脊髓，并使用虹膜剪脊髓切断神经根在他们的切入点，然后取出脊髓。 </li><li>转移与按病种付费其余椎骨充满冷学联第二培养皿。识别L4和根据它们的相对位置，以横突和坐骨神经L5的DRG。 注:维护坐骨神经完好无损将有助于确定L4和L5 DRG（坐骨神经从L4-6脊神经起源）。 </li><li>使用杜蒙钳和剪刀诺伊斯从周围结缔组织释放病种付费。保持神经根和脊髓神经连接到病种付费。 </li><li>用移液管的病种进入第三培养皿进一步解剖。 </li><li>根据解剖显微镜，小心地取出尽可能的周围神经外膜的第ËDRG。 <ol><li>使用诺伊斯弹簧剪刀剪一开口，其中背部和腹部的根加入DRG，并从DRG分离神经前根。使用杜蒙＃5镊子钝头举行外膜，并用另一细镊子从滚神经外膜的DRG。 </li><li>继续尽可能的外膜去除附着使用细镊子病种付费。 注意:良好的解剖DRG重要的是要获得在下一步骤良好的消化。 </li></ol></li></ol> 3. DRG消化<ol><li>转移DRG到记录室中。使DRG，其中神经根起源的面孔下来肯定的一面。 注意:以这种方式，大部分神经元访问。 </li><li>使用锚来稳定DRG。灌流DRG含氧脑脊液在通过用蠕动泵，它被放置在一个表旁边的记录钻机相连的塑料管以0.5ml / min的速率。 </li><li>等待30分钟，以使细胞恢复。通过CCD摄像机评估DRG的红外下差分接口对比在40倍物镜放大倍率的质量（IR-DIC）光学元件。 注:按病种付费好通常包含很多轮，以及对比，神经元卫星细胞在其表面所包围。 </li><li>消化的DRG的表面的一个小区域。 注意:玻璃补丁移液器置于吸移管座，这是小接头允许管被附连到玻璃吸管与一个气密密封。 <ol><li>通过个别小口径橡胶管的两片连接两个1毫升注射器移液器持有人。把充满胶原酶入吸管持有者之一和一个空玻璃吸管成其他一一玻璃吸管。 </li><li>使用显微操作的移液管的位置就在上面的DRG。然后，在显微镜下放大，相互碰撞轻轻2移液器的尖端放大枪头（理想的情况下，直径为5-的开口10微米）。 </li><li>移动填充有酶移液器靠近DRG的表面，并简要通过管连接支架和通过移动柱塞约0.5ml注射器施加正压到含有吸管胶原酶。 注意:在压力下，酶的从吸管该流程将略有放大DRG神经元，其可作为标志酶的应用之间的空间。 </li><li>后10至15分钟，观察到剩下的外膜的碎片时，施加轻柔负压空吸管吸走的碎屑。 注意:在这种方式中，神经元和周围卫星细胞会清楚暴露并准备补丁记录。 </li><li>丢弃这两个吸管放入锐器容器。 </li></ol></li></ol> 4.膜片钳记录<ol><li>放置电极溶液（ 见表3）在冰上，以防止降解。装满玻璃的补丁吸管过滤细胞内溶液（注射器过滤器，孔径:0.2微米），并放置在探头吸管保持器吸管。通过降低吸移管到浴溶液中之前位移约1ml柱塞施加到吸管轻柔正压通过5毫升注射器。 注意:这可以防止吸移管从被堵塞。 </li><li>选择将模式旋钮放大器上的"V-钳"模式，然后在软件中打开"膜测试"接口。靠拢在显微镜下检查目标神经元的吸管。 注意:在完整的DRG制备，卫星细胞的薄层封装每个神经。 </li><li>使用从吸管正压遍历卫星细胞层直至神经元和卫星细胞的周围层之间空间的突然放大观察。继续前进对神经元的吸管，直到"酒窝"是在神经元观察到。 注:与比较从解除关联神经元的录音，正压需要是稍微大，这将有助于穿透卫星细胞层和增加成功的修补的机会。 </li><li>降低正压力。其次，实现了千兆欧姆密封，轻轻吸气。 注意:在此方法中，记录的成功率是至少70％。 </li><li>如前面19所描述获得全细胞记录的配置。 <ol><li>简单地说，通过一个简短而强劲吸力穿透神经细胞膜。可替代地，当施加抽吸使用放大器上的"扎普"功能。 </li><li>一旦全细胞模式被建立，通过在放大器转动电容和电阻补偿旋钮补偿全细胞电容和串联电阻（Rs）。 注:RS通常是5-20MΩ。 </li><li>摒弃细胞如果Rs最初是大于30兆欧;或Rs中的记录过程中超过20％的变化。也测量静息膜电位;放弃的小区，如果静息膜电位是超过-50毫伏。 </li></ol></li><li>关闭"膜测试"窗口。通过转动模式旋钮放大器选择"我钳位正常"模式。 </li><li>点击"开放协议"的软件，选择并加载协议用于测量rheobase。点击"记录"开始录制。测量输入电阻和rheobase通过注入分级系列在100 Pa的步骤去极化电流检查神经元兴奋性。 <ol><li>再次单击"开放协议"，并选择用于测量膜门槛的协议。一个500毫秒的去极化电流斜坡（2,000 PA / S）将被注入到神经元。 </li><li>使用数据采集和分析软件（ 例如 Clampfit）根据制造商的说明来分析所记录的痕迹。使用该软件的稳态IV RELAT的基础上计算的输入电阻值（R in）交付的超极化电流时ionship。移动光标来衡量rheobase和膜的阈值。 注意:以诱导该AP所需的电流的幅度被定义为rheobase，以及用于诱导的AP的最低电压被定义为膜的阈值。 </li></ol></li><li>记录的配体诱导电流。 <ol><li>填与特异性激动剂吸管。 注意:在当前的报告中，我们使用100μM的谷氨酸和100μMAITC诱导分别由谷氨酸受体和TRPA1受体介导的电流。 </li><li>检查吸管，以确保里面没有气泡。放置在移液管固定器的吸移管。吸管保持器连接与连接到药物分配系统的管。 </li><li>使用操纵器中的神经元的50微米移动吸移管。设置药物分配系统的压力为1磅和持续时间为1秒。切换记录模式到电压钳，和在-70mV夹紧。简要地通过药物分配系统施加压力来记录一个药物诱导的电流。 </li></ol></li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+patch+clamp+recording+and+calcium+imaging+of+neurons+in+the+intact+dorsal+root+ganglion+in+rats.">An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats.</a> J Neurosci Methods. 173, 74-82 (2008).</li><li>Yagi, J., Sumino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+a+hyperpolarization-activated+current+by+clonidine+in+rat+dorsal+root+ganglion+neurons.">Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons.</a> J Neurophysiol. 80, 1094-1104 (1998).</li><li>Ma, C., Donnelly, D. F., LaMotte, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+and+functional+characterization+of+primary+somatic+neurons.">In vivo visualization and functional characterization of primary somatic neurons.</a> J Neurosci Methods. 191, 60-65 (2010).</li><li>Vit, J. P., Jasmin, L., Bhargava, A., Ohara, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+glial+cells+in+the+trigeminal+ganglion+as+a+determinant+of+orofacial+neuropathic+pain.">Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain.</a> Neuron Glia Biol. 2, 247-257 (2006).</li></ol>

The authors declare that they have no competing financial interests.

我们报告准备膜片钳研究整个病种付费的方法。有用于制备理想的检体的几个关键要素。首先，与附后根解剖的DRG是重要的。在此之后，神经外膜需要避免对神经元损害，同时要小心除去。最后，以暴露的神经元和其周围卫星细胞，有必要以消化剩余的结缔组织。从这里所描述的方法制备的成年大鼠完整的DRG将保持良好的生存力为6至8小时，并且可以在该期间稳定地记录。它们可用于诊断相关组?...

感觉是一个有机体的生存和健康的基石。刺激的传输依赖于起始于从初级感觉神经元轴突的周神经末梢的感觉通路。初级感觉神经元，与三叉神经的脑核外，位于三叉神经节和背根神经节（背根节）。他们担任的感官信息1看门人。在perikarial膜，正如在中央和外围终端，DRG神经元表达的受体和离子通道，例如谷氨酸受体，TNFα的受体，瞬时受体电位阳离子通道亚家族V部件1（TRPV1），钠通道等 2 -7。在perikarial膜的膜片钳记录可以理解许多这些受体和渠道遍布神经细胞的功能变化。 膜片钳记录技术是STU一个强大的工具垂死的通道或受体和大量的研究活动已被应用在背根神经节8-10这项技术进行的。在大多数研究中，DRG通过切割背侧支根和脊神经靠近神经节除去。切碎后，将神经节然后被放置在导致DRG神经元，然后可记录之前立即或培养记录数天的分解酶。不幸的是，DRG神经元的解离涉及接近胞体的必要干切断。一旦分离和金黄地鼠，DRG神经元发生在膜兴奋11,12的表型变化以及变化。通常围绕它们单个神经元的胞体和卫星细胞之间的接触的损失是可能有助于这些变化13。神经元和卫星细胞之间的串扰是生理条件缺一不可，并在适应patholog的iCal条件如那些导致顽固性疼痛14,15。这将是具有挑战性的，研究使用解离的DRG制备神经元和卫星细胞之间的相互作用。 完好的DRG，另一方面，提供接近体内条件。在过去的几年里，我们的实验室，以及一些其他基团，一直使用从成年大鼠完整的DRG以调查在慢性疼痛3-5,11,15-17相关联的不同条件的初级感觉神经元的变化。虽然在这些研究中所使用的技术是有些建立，步骤一步描述尚未公布。在本手稿中，我们描述了一个方便，快捷的方式准备完整的按病种付费和他们的膜片钳记录使用。

<table border="0" cellpadding="0" cellspacing="0" width="1016">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:252px;">Pentobarbital sodium</td>
			<td style="width:172px;">vortech Pharmaceuticals</td>
			<td style="width:108px;"></td>
			<td style="width:484px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>1 ml, 5 ml.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scalpel</td>
			<td>BD</td>
			<td></td>
			<td>size: 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayo straight scissor</td>
			<td>Fine Science Tools</td>
			<td>14010-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayo curved scissor</td>
			<td>Fine Science Tools</td>
			<td>14011-15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16021-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adson toothed forceps</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris Scissor</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Noyes spring scissor</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bone scissors</td>
			<td>Fine Science Tools</td>
			<td>16044-10</td>
			<td>Special for cutting the bones.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps: Dumont, Dumoxel Biologie #5</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>These have the fine tips that do not need sharpening when first purchased.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">periosteal elevator</td>
			<td>Sklar</td>
			<td>97-0530</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissection microscope</td>
			<td>WILD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Fisher brand</td>
			<td>13-711-5AM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish (10 cm)</td>
			<td>Pyrex</td>
			<td></td>
			<td>Glass petri dish can avoid damaging the tips of fine forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase (Liberase TM)</td>
			<td>Roche</td>
			<td>05-401-119-001</td>
			<td>dissolve at the concentration of 13 u/ml, aliquot into glass pipette. Avoid repeated freeze and thaw.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">filter</td>
			<td>Thermo scientific</td>
			<td>7232520</td>
			<td>Filter the internal solutions for patch clamp recording to avoid clog.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass pipette</td>
			<td>Sutter</td>
			<td>BF150-110-7.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anchor</td>
			<td>Havard apparatus</td>
			<td>64-0250</td>
			<td>stabilize the DRG to avoid drift.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peristaltic pump</td>
			<td>WPI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette puller</td>
			<td>Sutter</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular devices</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitizer</td>
			<td>Molecular devices</td>
			<td>1440D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>NIKON</td>
			<td>FN600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-manipulator</td>
			<td>Sutter</td>
			<td>MPC200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microinjection dispense system</td>
			<td>General Valve</td>
			<td>Picrospitzer II</td>
			<td>fast drug application system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbogen (95% O2, 5% CO2)</td>
			<td>Local Medical Gas supplier</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

patch clamp recordings on intact dorsal root ganglia from adult rats

从背根神经节膜片钳研究（病种付费）神经元增加了我们周围神经系统的理解。目前，大部分的录音是在解离的DRG神经元，这是大多数实验室标准制剂进行。神经元特性，然而，可以通过从在获取解离的神经元使用的酶消化所得轴索损伤改变。另外，解离的神经元制剂不能完全代表DRG的微环境，因为与环绕初级感觉神经元卫星细胞接触的损失是这种方法的不可避免的结果。为了克服在使用传统的分离DRG神经元膜片钳记录，本报告中的局限性，我们描述编写完整的按病种付费和个人的初级感觉神经元在体外进行膜片钳记录的方法。这种方法可以完整的DRG的快速和简单的准备， 在模仿体内通过保持与周围卫星细胞和基底膜相关的背根神经节的条件。此外，该方法从操作避免性轴索损伤和酶消化这种游离病种付费时。此体外制剂可以附加地用于研究初级感觉神经元和卫星细胞之间的相互作用。

图1显示了制备完整的DRG补丁记录的过程。 图1A示出椎板切除。 图1b后神经节的曝光和位置表示的L3，L4和L5的DRG以除去脊髓后附的神经根。然后L4和5的DRG仔细解剖并从椎骨释放。接着，将神经外膜，围绕DRG的透明膜被去除（黄色箭头， 图1D）。到神经外膜分离的最佳位置是通过那里的背侧和腹侧根部加入在DRG的部位，由<st...

Watch this Scientific Journal Video about Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats at JoVE.com

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

膜片钳记录的成年大鼠完整背根神经节

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

patch-clamp-recordings-on-intact-dorsal-root-ganglia-from-adult-rats

Research

JoVE Journal

Biochemistry

14.1K 次观看.  University of California, San Francisco. This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

视频: Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

开放补丁夹具移液器测量未搅拌边界层中的离子浓度: 流体流动对离子通道控制的启示

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

将新生儿小鼠心脏巨噬细胞移植到成年小鼠体内

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...