我们感谢为我们提供与离子通道的研究设施和良好的环境，加州大学戴维斯分校生理学与膜生物学部。

1。准备休息的人T淋巴细胞<ol><li>使用RosetteSep人类T细胞富集鸡尾酒和RosetteSep密度中等，净化T细胞从人体血液样本，根据制造商的指示。由此产生的细胞群，应含有95％的CD3 +静止T细胞。我们净化人类T淋巴细胞，从外周血中，从健康志愿者按照由加州大学戴维斯分校的内部审查委员会批准的协议采集的样本。 </li><li>隔离后，重新悬浮静止T细胞在细胞培养液中中含有谷氨酰胺和HEPES的RPMI - 1640培养基的密度在0.5 × 10 6细胞/ mL，辅以10％小牛血清，2％GlutaMAX，我，1％的RPMI 1640维生素溶液，1％的RPMI 1640氨基酸溶液，1％丙酮酸钠，0.03％β-巯基乙醇。 </li><li>放入细胞培养瓶中的细胞悬液，并保持在37 ° C的培养箱在5％的CO 2至24小时之前的实验。 </li></ol> 2。录音商会的制备<ol><li>以下是需要以准备录音商会：一个）硅Ø -环，b）轮，70％乙醇和蒸馏水H 2 Ø，彗星清洗25毫米盖玻片）混合Sylgard184，根据制造商的指示编写，和D）两个60 × 15毫米的培养皿中。 </li><li>地点〜0.5毫升混合Sylgard184成60x15毫米的培养皿。 </li><li> O形圈的下部浸入Sylgard184使用镊子。 </li><li>放置在一个干净的表面，如另一个培养皿中，O型环O形圈的边缘删除多余的Sylgard184。 </li><li>盖玻片上环和按下来。 </li><li>在85 ° C是40-60分钟或直到Sylgard184聚合的加热商会治愈Sylgard184。 </li><li>无尘埃的容器存放在录音室。 </li></ol> 3。制备补丁移液器<ol><li>在实验的当天，拉1〜2微米的提示使用玻璃毛细管的直径和吸管拉马的移液器。我们用长丝的高硼硅管（外径：1.5 mm和ID：1.10毫米）。在无灰尘的位置的商店移液器。 </li><li>大衣枪头Sylgard184或HIPEC R6101半导体防护涂层，按照标准程序9。 </li><li>轻轻地火抛光实验前的移液器。 </li></ol> 4。膜片钳安装准备<ol><li>配置并保存在控制你的膜片钳放大器软件gigaseal形成的宏。我们使用Windows XP和脉冲数据采集软件的支持下，EPC - 10放大器。我们设置的“设置”，“细胞”，和“全细胞”宏根据EPC - 10手动和所有的实验中使用它们。 </li><li>实验前准备洗澡的解决方案和表1中列出的吸管解决方案，并储存于4 ° C 1周。 </li></ol><table border="1"><tbody><tr><td>化学品</td><td colspan="3">沐浴解决方案（MM） </td><td>移液器解决方案（毫米） </td></tr><tr><td></td><td> ＃1 </td><td> ＃2 </td><td> ＃3 </td><td></td></tr></tbody><tbody><tr><td> CH 3 SO 3娜</td><td> 130 </td><td> 110 </td><td> 125 </td><td> - </td></tr><tr><td>氯化钠</td><td> 2 </td><td> 4 </td><td> 5 </td><td> - </td></tr><tr><td> Ca（OH）2的</td><td> - </td><td> 20 </td><td> - </td><td> - </td></tr><tr><td> MgCl 2的 </td><td> 3 </td><td> 1 </td><td> - </td><td> 5 </td></tr><tr><td> 硫酸镁 </td><td> - </td><td> - </td><td> - </td><td> 2 </td></tr><tr><td> HEPES </td><td> 10 </td><td> 10 </td><td> 10 </td><td> 15 </td></tr><tr><td> HEDTA </td><td> - </td><td></td><td> 10 </td><td> - </td></tr><tr><td> EDTA </td><td> - </td><td> - </td><td> 1 </td><td> - </td></tr><tr><td> CS - Aspartate </td><td> - </td><td> - </td><td> - </td><td> 125 </td></tr><tr><td> BAPTA </td><td> - </td><td> - </td><td> - </td><td> 12 </td></tr><tr><td>血糖</td><td> 10 </td><td> 10 </td><td> 10 </td><td> - </td></tr></tbody></table> 表1。全细胞CARC当前的录音解决方案。 均购自Sigma - Aldrich公司，圣路易斯，密苏里州的所有化学品。 <ol start="3"><li>确定液体的交界处之间补丁吸管的解决方案，并每个液10的电位（ LJ）。保存在适当的地方使用采集软件的LJ值。例如，脉冲程序中插入的“LJ”控制的“放大器”窗口中的LJ值。 </li><li>配置和刺激的协议，在适当的地方采集软件之前的实验，如图1所示，保存。在脉冲程序，我们在“脉冲产生”窗口中设置了收购协议。 <img src="/files/ftp_upload/2346/2364fig1.jpg" alt="图1" /> 图1。刺激协议从-120到100毫伏的持续时间在50毫秒的电压斜坡是每0.2 - 2秒（5 - 0.5赫兹） 。斜路之间的电位（V H）维持在30毫伏。 </li></ol> 5。显微镜舞台上安装的细胞<ol><li>大衣与聚- L -赖氨酸〜20分钟，洗净与H 2 O，然后在电镀前的细胞与汉克的平衡盐溶液（HBSS）的录音室。 </li><li>板500μL进入会议厅和20分钟的孵育的细胞悬液，含0.2-0.5 × 10 6细胞在37 ° C 。 </li><li>凭借一腔持有人的贴壁细胞的录音室。我们使用一个特制的商会持有人，基地支持盖玻片的底部，而上面的部分轻轻地对盖玻片上方的印刷机。 </li><li>放置在倒置显微镜下阶段的商会持有。 </li><li>细胞集中在透射光。我们使用了40X的油浸物镜。 </li><li>放入浴缸的Ag / AgCl参比电极。 </li><li>将多管，重力驱动灌注系统。流入管的尖端应放在45 °角，以盖玻片接近视野。 </li><li>吸痰管流入管的尖端对面的位置。 </li><li>灌注室与磷酸盐缓冲液（PBS）或HBSS洗去细胞培养基和松散贴壁细胞。 </li></ol> 6。 CRAC通道电流记录<ol><li>找到一个牢固地附着在盖玻片的细胞。我们通常会选择一个平均大小的圆形细胞，具有光滑的外表面。 </li><li>灌注与Ca 2 +，3 mM的Mg 2 +的含液＃1（表1）含有0.5-1块SERCA泵为8-10分钟微米毒胡萝卜素的录音室。我们执行这一步gigaseal形成之前消耗存储。 </li><li>使用的Eppendorf microloader和一个P - 20位移移液器，填充液（见表1）补丁吸管和放大器探头插入吸管持有的补丁吸管。的Ca 2 +螯合剂BAPTA液，被动地消耗商店和减少的Ca 2 +依赖的CRAC通道的失活。 </li><li>应用补丁吸管内再入浴少量的正压（〜3英寸的H 2 O）。我们使用一个特制的压力吸气的设备连接到加压的空气和真空线，以创建补丁吸管内的积极或消极的压力。 </li><li>通过显微镜下的可视化控制，降低到洗澡的补丁吸管。在“示波器”窗口中，你应该看到一个电流流经吸管。在电流幅值的一个阶梯状的变化应引起一个小幅度的电压脉冲，通过预设的宏中的应用（测试脉冲）。这时，你应该能够确定吸液管电阻值。我们移液器的电阻通常为5-6MΩ的。 </li><li>纠正“抵消”吸管和参比电极之间产生的潜在。 </li><li>通过显微镜的视觉控制下，把枪头更接近，直到它触及到细胞的细胞膜。 </li><li>应用补丁吸管内的负压（20-40英寸的H 2 O） 。 </li><li>监视器在“示波器”窗口gigaseal形成。你会看到目前的幅度，测试脉冲，减少和吸液管电阻增加值&gt; 5GΩ的诱导。在FE的通常gigaseal形式瓦特秒，但它可能需要1-2分钟，以达到稳定gigaseal。 </li><li>补偿吸管电容（“快”）。 </li><li>申请额外的负面压力，用20毫升注射器打破补丁膜。 </li><li>设置到30毫伏电位。了积极的控股潜力，有助于防止钙依赖性的CRAC通道的失活。 </li><li>补偿细胞膜电容（“慢”）;保存或记录的C -慢的价值。 </li><li>检查访问电阻“R S”值。在我们的实验中，R S通常是7-20兆欧。 </li><li>开始申请一个0.5赫兹的频率在Ca 2 +免费3毫米Mg 2 +的含液＃1（图1）一系列电压斜。在这个解决方案，机房空调的电流是可以忽略不计小的“泄漏”目前由于Mg 2 +的通透性差的CRAC通道相比。保存和使用沐浴液＃1“泄漏”的电流记录在未来的分析当前的痕迹。 “泄漏”目前在-100 mV的绝对幅度应小于或等于5 pA的。如果细胞是“漏”，停止对使用一个新的补丁吸管和一个新的单元格的实验，并开始。 </li><li>要记录的Ca 2 +电流通过的CRAC通道（CA - CRAC）， 通过切换灌注阀，取代20 毫米的Ca 2 +含液＃2（表1）的Ca 2 +免费3毫米Mg 2 +的含液＃1 。这允许的Ca 2 +通过的CRAC通道的流量，产生内向电流。在“示波器”窗口中，你会看到内向整流我CA机房空调（图2）的发展。目前在负电压的振幅将继续增加约1分钟的Ca 2 +依赖的CRAC电流程增强。 </li><li>增加刺激频率5 Hz，这是必要的记录通过的CRAC通道的瞬态钠电流电压斜。 </li><li>替换20毫米的Ca 2 +含Na +的含二价阳离子免费的解决方案＃3（见表1）通过切换灌注阀液＃2。在“示波器”窗口中，你会看到一个振幅较大的瞬态发展， 内向整流娜+，目前通过的CRAC通道（我娜的CRAC图2）。 </li><li>一个可能适用于20毫米的Ca 2 +含液含有的实验记录“泄漏”目前结束在1-5微米镧+＃2。然而，我们没有发现这种方法非常有用，因为“泄漏电流的变化，”随着时间的推移，它可能在实验结束后成为更大或更小。我们宁愿采取记录中的Ca 2 +免费3毫米的电流镁2 +含液为“泄漏”目前的实验开始于＃1 。 </li><li>保存记录作进一步分析当前的痕迹。 </li></ol> 7。数据分析<ol><li>检索到分析软件保存当前记录。我们使用脉冲程序进行分析。 </li><li>测量电压斜在开始和结束目前的幅度。我们通常在-100 mV和100 mV的“示波器”脉冲程序的窗口在使用游标对测量的电流幅度。 </li><li>成作进一​​步的分析和图形显示的图形程序中导出电流幅度值。我们使用原产科学绘图和分析软件，第7版。 </li></ol> 8。代表性的成果<img src="/files/ftp_upload/2346/2346fig2.jpg" alt="图2" /> 图2。机房空调在休息的人类T细胞的电流 （A），当然在全细胞电压钳在-100 mV的（实心圆）和100毫伏（空心圆）的配置记录的CRAC电流。 gigaseal形成之前，细胞孵育〜10分钟，在Ca 2 +免费的3 毫米MG 2 +含液＃1含0.5微米毒胡萝卜素。 CA的突破后，洗澡的解决方案相继被应用如下：2 + 3 毫米MG 2 +含液＃1（0钙3毫克），20毫米的Ca 2 +含液＃2 （20钙），二价阳离子无液液＃2（20钙）＃3（DVF），其次是。一系列如在图1所示的电压斜的细胞受到刺激。斜道的频率为5赫兹液＃3（DVF）在所有其他解决方案和0.5赫兹。请注意应用20 毫米的Ca 2 +含液＃2和我那机房空调的快速瞬态发展DVF液＃3后发展缓慢， 我钙的 CRAC 。 （二），代表当前的痕迹记录在Ca 2 +无3 mM的镁2 +含液＃1（在电压斜“泄漏”），20毫米的Ca 2 +含液＃2（20钙），液＃3（DVF）。 （C，D），我CA的CRAC（C）和我娜的CRAC（四）获得减去“泄漏”电流电压斜的Ca 2 +含浴期间录得20毫米的电流的电流-电压关系解决方案＃2（20钙），沐浴液＃3（DVF）面板（b）所示。

<ol>
	<li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat Rev Drug Discov. 9, 399-410 (2010).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRAC+channelopathies.">CRAC channelopathies.</a> Pflugers Arch. , (2010).</li><li>Lewis, R. S., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+inactivation+of+depletion-activated+calcium+current+(ICRAC)+due+to+local+calcium+feedback.">Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.</a> J Gen Physiol. 105, 209-226 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium-dependent+inactivation+of+depletion-activated+calcium+current.+Store-dependent+and+-independent+mechanisms.">Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms.</a> J Biol Chem. 270, 14445-1451 (1995).</li><li>Zweifach, A., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+potentiation+of+store-operated+calcium+channels+in+T+lymphocytes.">Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.</a> J Gen Physiol. 107, 597-610 (1996).</li><li>Prakriya, M., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+currents+through+store-operated+CRAC+channels+and+Mg2+-inhibited+cation+(MIC)+channels.">Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels.</a> J Gen Physiol. 119, 487-507 (2002).</li><li>Fomina, A. F., Fanger, C. M., Kozak, J. A., Cahalan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+channel+properties+and+regulated+expression+of+Ca(2+)+release-activated+Ca(2+)+(CRAC)+channels+in+human+T+cells.">Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.</a> J Cell Biol. 150, 1435-1444 (2000).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-Channel Recording. , (1995).</li><li>Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+for+liquid+junction+potentials+in+patch+clamp+experiments.">Correction for liquid junction potentials in patch clamp experiments.</a> Methods Enzymol. 207, 123-1231 (1992).</li></ol>

人类T细胞在休息的CRAC电流的电生理调查是一项具有挑战性的任务，因为在这些细胞中的内源性的CRAC的电流幅值小，由于小单元尺寸（休息人体T细胞直径在5-8微米的范围内）。在这里，我们现在一步一步的程序，以可靠地记录在休息的人类T淋巴细胞从外周血单个核细胞中分离的CRAC电流。这项技术使我们能够探讨在静息T细胞的CRAC通道的生理学和功能表达，以便更好地了解正常和病理的免疫细胞?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RosetteSep Human T Cell Enrichment Cocktail</td>
<td>&nbsp;</td>
<td>StemCell Technologies, Vancouver, BC, Canada</td>
<td>15061</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep Density Medium</td>
<td>&nbsp;</td>
<td>StemCell Technologies </td>
<td>15705</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-in 1640 medium w/glutamine/HEPES</td>
<td>&nbsp;</td>
<td>Fisher, Waltham, MA</td>
<td>SH3025501</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum</td>
<td>&nbsp;</td>
<td>Omega Scientific, Tarzana, CA</td>
<td>FB-01</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX-I (100X solution)</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>35050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 vitamin solution (100X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>7256</td>
<td>&nbsp;</td>
<tr>
<td>1640 amino acids solution (50X)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R7131</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>Inositol trisphosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>19766</td>
<td>&nbsp;</td>
<tr>
<td>BAPTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A4926</td>
<td>&nbsp;</td>
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<td>Poly-L-Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2636</td>
<td>&nbsp;</td>
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<td>Lanthanum Chloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>262072</td>
<td>&nbsp;</td>
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<td>Thapsigargin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>586005</td>
<td>&nbsp;</td>
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<td>Sylgard 184 Silicon Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>3097358-1004</td>
<td>&nbsp;</td>
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<td>HIPEC R6101 Semiconductor Protective Coating</td>
<td>&nbsp;</td>
<td>Dow Corning, Midland, MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>63-500 Series High-Performance Vibration Isolation Lab Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing, Peabody, MA</td>
<td>63-540</td>
<td>&nbsp;</td>
<tr>
<td>EPC 10 patch clamp amplifier with headstage</td>
<td>&nbsp;</td>
<td>HEKA Instruments, Bellmore, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator </td>
<td>&nbsp;</td>
<td>Sutter Instrument, Novato, CA </td>
<td>MP-285</td>
<td>&nbsp;</td>
<tr>
<td>Olympus 1X71 Inverted microscope with 40x oil immersion objective</td>
<td>&nbsp;</td>
<td>Olympus America, Center Valley, PA</td>
<td>1X71</td>
<td>&nbsp;</td>
<tr>
<td>Windows Computer</td>
<td>&nbsp;</td>
<td>Dell </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pulse software</td>
<td>&nbsp;</td>
<td>HEKA Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Origin Scientific Graphing and Analysis Software</td>
<td>&nbsp;</td>
<td>OriginLab, Northampton, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Patch pipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass with filament (O.D.: 1.5mm and I.D.: 1.10mm</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF150-110-7.5</td>
<td>&nbsp;</td>
<tr>
<td>Narashige's Microforge</td>
<td>&nbsp;</td>
<td>Tritech Research, Los Angeles, CA</td>
<td>MF-830</td>
<td>&nbsp;</td>
<tr>
<td>Silicon O-rings </td>
<td>&nbsp;</td>
<td>McMASTER-CARR, Santa Fe Springs, CA</td>
<td>111 S70</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips 25 mm </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>12-545-102 25 mm 25CIR.-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recording of calcium release-activated calcium (crac) currents in human t lymphocytes

T淋巴细胞，耗尽的Ca 2 +从细胞内Ca 2 +存储信息激活质膜钙离子通道，被称为钙释放激活钙（CRAC）通道。 CRAC通道中的T细胞增殖和基因表达调控中发挥重要作用。 T细胞异常的CRAC通道的功能已被链接到重症联合免疫缺陷和自身免疫性疾病1，2。在人类T细胞研究的CRAC通道功能调节正常的免疫反应，可能会发现新的分子机制，并揭开有关人类疾病的原因。膜电流的电生理记录功能通道特性及其调控提供最准确的评估。然而，在正常的人体T细胞的CRAC电流测量，人类白血病T细胞株，Jurkat T细胞的CRAC通道电流的电生理评估是第一次执行超过20年，前3仍然是一个具有挑战性的任务。在录音中正常T细胞的CRAC通道电流的困难加剧的事实，血源性T淋巴细胞Jurkat T细胞的大小比要小得多，因此，全细胞内源性的CRAC电流幅度非常低。在这里，我们给一步一步的过程，我们经常使用记录的 Ca 2 +或Na +通过在休息从健康志愿者的外周血中分离的人类T细胞的CRAC通道的电流。这里描述的方法记录在Jurkat T细胞的CRAC电流激活人体T细胞4-8所使用的程序获得通过。

Watch this Scientific Journal Video about Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes at JoVE.com

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

钙离子释放活化钙全细胞膜片钳记录（CRAC）外周血单核细胞衍生的人类T淋巴细胞中的电流，我们提供了一步一步的协议。

在人T淋巴细胞的全细胞钙释放激活的钙记录（CRAC）的电流

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

whole-cell-recording-calcium-release-activated-calcium-crac-currents

Research

JoVE Journal

Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

16.0K 次观看.  University of California, Davis. 钙离子释放活化钙全细胞膜片钳记录（CRAC）外周血单核细胞衍生的人类T淋巴细胞中的电流，我们提供了一步一步的协议。

视频: Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

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, ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...