作者想感谢格伦斯洛克姆（威斯康星医学院）和科琳A.拉文（尼康仪器公司）与显微镜实验优秀的技术援助。格雷戈里·布拉斯是公认的手稿校对至关重要。这项研究是支持的健康补助HL108880和美国糖尿病协会的国家机构给予1-15-BS-172（AS到），以及本J.里普斯研究奖学金由美国肾脏病学会（至DVI）。

动物使用和福利要坚持NIH指南实验动物以下机构动物护理和使用委员会（IACUC）审查和批准协议的管理和使用。 1.肾冲洗<ol><li>使用8〜12周龄雄性大鼠（建议是只SD菌株，但不同年龄和性别的其他菌株可以适当变化来使用）。 </li><li>根据由IACUC协议所允许的过程麻醉动物;监测麻醉深度，检查动物。将在1.3实施手术的详细描述- 1.8可在Ilatovskaya 等人找到7。 </li><li>适当麻醉后，将动物在一个温度控制的手术台，使腹部中线切口（最多3英寸长），并揭示了腔静脉和主动脉。 </li><li>将周围结扎腹腔及肠系膜上动脉和腹主动脉高于;不结扎。 </li><li>钝解剖低于肾动脉腹主动脉，并把周围的两份结扎，但不结扎，然后夹住上述结扎主动脉和领带底线。 </li><li>导尿用聚乙烯PE50管（连接于注射器泵充满PBS）中的钳下面主动脉和固定与所述第二绑带的导管;除去夹具，打开泵，并结扎主动脉和肠系膜腹腔动脉。迅速使切口肾静脉，以减轻压力。 </li><li>注入与预冷却的PBS主动脉为2或3分钟以6ml / min的速率。 </li><li>停止灌注，消费税和7解封肾脏，并把它们在冰上PBS液。根据经批准的协议IACUC安乐死的动物。 </li></ol> 2.分离的大鼠肾小球<ol><li>制备将30ml 5％的BSA的新鲜溶液中的RPMI 1640。 </li><li>用刀片和剪刀，隔离双肾皮质，然后剁碎直到均匀。这个程序已经较早7描述。 </li><li>通过100目的不锈钢筛推在先前步骤剁碎的组织使用刮刀（预先浸泡在5％BSA / RPMI溶液）。收集流过并通过重力允许流动通过穿过一个140目筛。 </li><li>过滤流通从140目筛收集的使用预浸渍200目筛网，弃去滤液，并用10筛的顶部 - 将15ml制备的BSA / RPMI溶液来收集沉淀的肾小球筛。 </li><li>把含有在冰上肾小球BSA / RPMI溶液在一个15毫升管并让肾小球沉淀在试管底部达20分钟。肾小球集中在管的底部将被看得很清楚。除去多余的溶液，留下约2毫升管中。 </li></ol> 3.单通道膜片钳萨尔瓦多ectrophysiology <ol><li>准备5×5mm的玻璃盖片与MW 70000涂层他们 - 150000聚ʟ赖氨酸，​​让干。每护罩玻璃使用约30微升的0.01％的无菌过滤的溶液中的水。 </li><li>预热实验溶液至室温，并填充膜片钳腔和吸管。对于TRPC通道监控，使用沐浴液，在MM:126氯化钠，氯化钙1 2，10 HEPES，2 氯化镁 ，10葡萄糖，pH值7.4;吸管:126氯化钠，氯化钙1.5 2，10 HEPES，10葡萄糖; pH为7.4。 <ol><li>添加抑制剂的吸移管溶液来阻断内源性通道，这是不相关的研究（建议的活动有:100μM的尼氟酸或DIDS（方框钙激活Cl -的信道），10mM的TEA（以抑制大电导钙离子-依赖性K +通道），10nM的iberiotoxin（阻止的钙激活K +通道），10μM尼卡地平（方框N型Ca2+直接膜片钳实验前通道））。 </li></ol></li><li>轻轻地混合含肾小球溶液，然后应用大约50微升到聚ʟ赖氨酸包被的玻璃盖片。让肾小球附加约5分钟。 </li><li>移动的玻璃碎片与肾小球的膜片钳室预先填充的浴溶液;灌注腔室以3毫升/分钟进行1分钟的速率，以保证在除去未附着的肾小球。 </li><li>进行常规的膜片钳实验中一个细胞贴附模式7。用玻璃移液管（7 - 10 M吸液管电阻）形成吸管和通过施加轻柔抽吸（附连到分离的肾小球的表面上的足细胞吸管一个足细胞膜之间的高电阻密封示于图2中 ，上左边）。 </li><li>对于细胞贴附测量，由一个八极贝塞尔滤波器低通的电流，在300赫兹。 </li><li> ü本身分离肾小球在膜片钳实验长达4 - 6小时。置于冰上股票肾小球部分。 </li></ol>在足细胞内钙离子浓度成比例4.共聚焦荧光测量<ol><li>将500微升肾小球分数（在2.5描述）的0.5毫升的锥形管，并补充钙染料的Fura红，AM和荧光 - 4，AM。使用2毫米和1mM储备浓度的Fura红，AM和的Fluo-4，AM，分别为（储存在-20℃下，溶解在DMSO中），并使用2.5微升每种染料为500微升肾小球馏分。立即加入染料后用铝箔覆盖的管。 </li><li>地方管在旋转摇动器至少20分钟到1小时，在室温。 注意:药理剂可在此步骤中加入。 </li><li>制备的玻璃盖玻片，用聚ʟ赖氨酸覆盖它们，并允许使用加热板设定为70℃的干燥。 </li><li>一旦钙染料的负载量为COMPLET即，应用100微升含有溶液的聚ʟ赖氨酸包被的盖玻片的肾小球，让他们粘在表面上为约5分钟。安装肾小球附着盖玻片到成像室中，并灌注同浴溶液（含有（以mM计）:145氯化钠，4.5氯化钾，2的MgCl 2，10 HEPES，pH值7.35）以3ml / min的速率以除去未连接的肾小球和剩余的染料。 </li><li>设置在共聚焦激光扫描显微镜488nm的激发波长和发射滤光片（25分之525和二十五分之六百五，纳米为的Fluo-4和的Fura红分别）。成像软件设定为所希望的频率和分辨率。 </li><li>发现在明肾小球，然后打开荧光信号的检测。调节激光的强度为每个染料，以避免信号的饱和度。与直接连接到玻璃中的足细胞选择焦平面。这响应于药物最小化由于肾小球的萎缩的效果应用。仔细检查选择的肾小球很好地附着在玻璃; </li><li>开始成像选择的焦平面（取为512×512的图像与频率设定为4秒来可视化快的Ca 2+瞬时变化。使用60X / NA的1.4或类似物镜，用于高分辨率图像），应用药物的兴趣，并记录的响应。 <ol><li>选择期望的焦平面（如靠近玻璃尽可能的表面上，以确保肾小球的表面上足细胞的成像）。荧光检查的Fluo4和FuraRed渠道的力度，并确保肾小球清楚地看到在明。 </li><li>启动成像。应用的任何药物之前，基线荧光记录至少1分钟，以确保该信号是稳定的（没有突然尖峰或衰落的信号）。 </li><li>应用所需的药物用微量的帮助;要小心，并确保药物是能够扩散良好，达到glomer汗国。混合槽液轻轻如果需要的话，监视选定焦平面并检查它没动，因为药物应用失焦。 </li><li>记录为Fluo4和FuraRed信号的荧光强度的变化。确保记录足够长时，通过等待直到信号达到平台期的水平或达到峰值，然后返回到基线。如果需要的话执行解变化或加入任何其它药物。 </li><li>停止录制，并保存在文件中的软件的本机格式。 </li></ol></li></ol>对于钙测量5图像分析<ol><li>与搭载ND实用插件，允许进口的原生ND2格式图像ImageJ的开源软件进行图像分析。 </li><li>导入图像序列;确保分裂渠道和使用hyperstack灰度模式。 </li><li>按照分析→工具→投资回报率经理路径在ImageJ的软件邻笔投资回报率管理器窗​​口。选择感兴趣的（足细胞），使用椭圆选择工具和"添加（吨）"中的投资回报率管理器功能几个区域。当最后的投资回报率，选择在后台区域;保存选定的ROI（更多→保存）。 </li><li>突出显示包含选择用于分析所述信道的窗口。使用更多→多功能测量功能的投资回报率经理，标志着在弹出的对话"一排每片"复选框，然后单击确定。结果将显示在可设置在结果窗口中的格式:进入菜单选项结果→设置测量，选择"平均灰度值"，并计算每个投资回报率，这将显示在像素强度值结果窗口中的每个单独的投资回报率列。 </li><li>复制所测量的ROI强度值的每个信道（的Fluo-4和的Fura红）到优选的数据分析软件;从每个DAT减去背景强度值一个点。 </li><li>对于每个时间点计算出的Fluo-4的强度来的Fura红信道的比率。剧情分散/的钙瞬变每个ROI线点时间的变化。计算平均/ SE值选定肾小球。 注意:时间列应根据在4.5选定的成像频率来设置。 </li></ol> 6.细胞内钙离子浓度计算使用荧光 - 4荧光信号<ol><li>取肾小球的样品，并执行实验方案直到步骤4.7。记录的背景荧光后添加离子霉素（终浓度在浴室应10μM）向浴溶液中，并记录荧光强度的增加。一旦强度达到其最大值和衰减开始，添加的MnCl 2（最终浓度应为5毫米）以猝灭荧光8。 </li><li>分析在6.1中获得的数据。根据获得的荧光 - 4信号ROI强度值复制以在5.1中描述的协议 - 5.5通过优选分析软件。 </li><li>计算出细胞内钙离子浓度（纳米）在使用公式相应的投资回报率足: <img alt="式（1）" src="/files/ftp_upload/52850/52850equation1.jpg" /> 其中K d是预先确定的解离常数的Fluo-4（345纳米），F是强度在您正在计算中的钙浓度为（基线）的时间点，和F 分钟和F 最大值是在强度值点的最大钙负荷（洛诺霉素应用后）和荧光（带有的MnCl 2），骤冷后，分别（ 见图3）。 </li></ol>

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作者什么都没有透露。

这里所描述的方法允许的钙处理由啮齿类肾小球的足细胞的分析。这种技术允许应用膜片钳单通道电生理和荧光比率共焦成像。然而，这两种方法可以单独使用，在他们自己的。该协议有几个相对简单的步骤，包括:1）肾脏冲洗; 2）肾小球通过差筛分分离; 3）进行膜片钳电生理实验，或用荧光钙标记染料更改胞内钙的比例共焦成像的肾小球的孵化。 为了分离肾小球，基于Gloy改性协议等人 5被使用。差动筛分的当前技术的主要优点在于，它产生肾小球剥去肾小囊，并因此不需要复杂的和费时的步骤手动拆封。多数在隔离部分肾小球进行解封装，但是研究者必须认识到，有封装肾小球了。确保剥离的肾小囊的肾小球成象是很重要的，因为日本防止药物到达足。正如在图2中，解封装肾小球具有粗糙表面具有独特毛细血管和足细胞体，而封装肾小球出现光滑和圆形。此外，封装Fluo4和FuraRed加载肾小球发出那么强烈的荧光信号进行比较，以解封装的。 大鼠肾小球获得的分数是95％纯的（与近端小管的痕迹）和如果需要，可以方便地用于蛋白质印迹或其他分子生物学应用。它是非常重要的是，在此制备足细胞附着于毛细管，保留完整脚过程和功能的一部分的天然肾小球过滤装置的机械。钙处理由足细胞的机制有助于在肾并发症的这类疾病如糖尿病或高血压9-12的发展和预防的重要的调节作用。足细胞显著的渗透性屏障贡献，其证据是急性损伤改变足细胞形态和结构，并可能导致蛋白尿2,3,13-15的事实。因此，它是非常重要的，观察钙流入的响应中，这些细胞对药物，这可以很容易地筛选在此制备。应当理解的是，如果研究人员正在探测足细胞在病理状态，例如，高血压或糖尿病，这通常会导致蛋白尿和肾小球损伤，肾小球的产率可能会高于健康动物的低纯度。它建议始终监视肾小球馏分在分离过程，以避免损失的每个阶段组织。在一些情况下，从老化或太幼小动物严重患病肾小球或肾小球可能需要隔离处理的个别调整，包括较大/较小目筛的选择。 我们已经采用这种方法最初确定的钙TRPC通道中足细胞16抑制的非类固醇消炎药的功效。下面的这些研究7,16-22我们采用上述技术，建立控制功能足几个关键机制。例如，我们已经描述了在P2（嘌呤）受体的轮廓在大鼠的足细胞和定义钙流入在小鼠肾小球的调节由血管紧张素Ⅱ。如在后面的原稿20描述的，该协议可以适于执行类似的研究，不仅在大鼠，而且在其他物种，例如小鼠。比例钙荧光成像（与使用两种荧光染料 - 的Fluo-4和的Fura红）保证了数据的额外的可靠性，并且，如果需要钙成像可以与其他荧光染料结合，以监测足细胞内变化的其他物质。例如，我们已经使用这种方法来测量一氧化氮与DAF-FM染料的帮助。膜片钳电生理学可单独使用或（设置允许）与钙成像组合。除了钙通道，该技术允许记录其他离子通道的活性。此外，使用转染和siRNA，可以扩大应用更加的区域，如最近已表明通过干燥机博士的研究小组23。 常规荧光显微镜在这里可以用来代替难以负担共焦成像。然而，研究人员应该意识到的宽视场荧光显微镜有效灵敏度通常受限于离焦的光。这（相比于共聚焦IMAGING）等原因引起的局限性无法产生单一的投资回报率（足）或其足突的高分辨率图像。此外，宽视场显微镜复杂识别足细胞，因为它是硬的肾小球和深部组织的表面之间进行区分。截至目前各种荧光共焦成像的商业可用性要好得多，并迅速扩张。另外，萤光的研究可以很容易地结合的电安装可同时提供细胞内钙离子和单声道录音活动。肾小球足细胞过滤轮单色和中性密度滤镜设置的Fura2-AM装载被广泛使用，但并不总是适合质量高分辨率成像。 还应当指出的是，分离的，功能性肾小球能够改变响应于生理刺激它的体积。研究人员应该意识到这一点的同时进行的实验中，并小心地选择在共聚焦研究焦平面，以避免失焦，并在选择的新颖的药物，以测试非预期收缩或松弛进行控制实验。 这种技术提供了一个独特的机会来监视在单离子通道和改变钙流入个体细胞水平的药理学治疗或其他操作后，在各种啮齿类背景。这种技术还可以用于在人类肾脏疾病的研究作为修正协议可以被应用到从肾脏活检，这无疑将提供非常有价值的信息获得的组织。此外，钙离子浓度的测量可配对的电生理膜片钳实验是实时的，其可以提供更深入的和机械的洞察钙的调节在正常和病理条件下的足细胞内处理。

肾脏维持自我平衡各种物质和的方式，确定总血压调节血容量。干扰在肾过滤，重吸收或分泌导致或伴随的病理状态，从高血糖或低血压结束末期肾病，最终需要肾移植。肾过滤单元（肾小球）由三层-毛细血管内皮，基底膜和上皮细胞的单细胞层-足细胞，从而起到的狭缝隔膜的完整性和功能1的维持起主要作用。功能障碍的选择性渗透肾小球过滤导致大分子，​​如蛋白的尿中丢失。各种试剂可以影响足细胞和它们的足突，这确定肾小球滤过屏障的完整性的结构。 足细胞都参与了格莱姆教授的维护eruli过​​滤功能。它已经确定，不当钙处理由足细胞导致细胞损伤和起着各种形式肾病2,3的发展中起重要作用。因此，一个模型，允许对细胞内钙离子浓度的变化直接测量将有助于对足细胞功能研究的发展。孤立肾小球在许多研究，包括白蛋白反射系数测量变化4，并在全细胞电生理膜片钳测量5,6-积分蜂窝电流的评估以前使用。在本论文中，我们描述的协议，该协议允许研究者测量响应于药理学试剂的应用细胞内钙离子浓度的变化，估算出细胞内钙的基础水平，和评估个体的钙离子通道的活性。 Ratometric钙离子浓度的测量和膜片钳electrophysiology分别用来确定足细胞和信道活动中的变化，在细胞内钙离子浓度。

<table><tbody><tr><td>Fluo4 AM</td><td>Life Technologies</td><td>F14217</td><td>500 &amp;micro;l in DMSO</td></tr><tr><td>FuraRed AM</td><td>Life Technologies</td><td>F-3020</td><td></td></tr><tr><td>Poly-&amp;#671;-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Pluronic acid</td><td>Sigma-Aldrich</td><td>F-68</td><td>solution</td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I3909-1ML</td><td></td></tr><tr><td>Tube rotator</td><td>Miltenyi Biotec GmbH</td><td>130-090-753</td><td>Germany</td></tr><tr><td>Nikon confocal microscope (inverted)</td><td>Nikon</td><td>Nikon A1R</td><td>Laser exitation 488 nm. Emission filters 500-550 nm and 570-620 nm</td></tr><tr><td>Objective</td><td>Nikon</td><td>Plan Apo 60x/NA 1.4 Oil</td><td></td></tr><tr><td>Cover Glass</td><td>Thermo Scientific</td><td>6661B52</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td></td><td>Silicone Compound</td></tr><tr><td>Software</td><td>Nikon</td><td>Nikon NIS-Elements</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data acquisition system</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Low pass filter</td><td>Warner Instruments</td><td>LPF-8</td><td>8 pole Bessel</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150F-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>Japan</td></tr><tr><td>Motorized micromanipulator</td><td>Sutter Instrument Co</td><td>MP-225</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Microvibration isolation table</td><td>TMC</td><td></td><td>equipped with Faraday cage</td></tr><tr><td>Multichannel valve perfusion system</td><td>AutoMake Scientific</td><td>Valve Bank II</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10.2</td><td></td></tr><tr><td>Nicardipine</td><td>Sigma-Aldrich</td><td>N7510</td><td></td></tr><tr><td>Iberiotoxin</td><td>Sigma</td><td>I5904-5UG</td><td></td></tr><tr><td>Niflumic acid</td><td>Sigma-Aldrich</td><td>N0630</td><td></td></tr><tr><td>DIDS</td><td>Sigma-Aldrich</td><td>D3514-25MG</td><td></td></tr><tr><td>TEA chloride</td><td>Tocris</td><td>T2265</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11835030</td><td>without antibiotics</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A8327</td><td>30% albumin solution</td></tr><tr><td>Temperature controlled surgical table</td><td>MCW core</td><td></td><td>for rodents</td></tr><tr><td>Steel sieves:</td><td></td><td></td><td>#100 (150 &amp;mu;m), 140 (106 &amp;mu;m)</td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO200</td><td>Fisher Sci</td><td>50-871-316</td><td></td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO270</td><td>Fisher Sci</td><td>50-871-318</td><td></td></tr><tr><td>Gilson, Inc SIEVE 3 SS FH NO400</td><td>Fisher Sci</td><td>50-871-320</td><td></td></tr><tr><td>mesh 200</td><td>Sigma-Aldrich</td><td>s4145</td><td>screen for CD-1</td></tr><tr><td>Binocular microscope</td><td>Nikon</td><td>Eclipse TS100</td><td></td></tr><tr><td>Binocular microscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Syringe pump-based perfusion system</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Polyethylene tubing</td><td>Sigma-Aldrich</td><td>PE50</td><td></td></tr><tr><td>Isofluorane anesthesia</td><td><a href="http://www.vetequip.com/" target="_blank"><img src="/img/new_window.png" /> VetEquip, Inc.</a></td><td>911103</td><td></td></tr><tr><td>Other basic reagents</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

这里我们讨论测量在足细胞中的钙水平急性改变的问题。 图1显示了实验方案设计，以便在新鲜的足细胞进行高分辨率实时荧光共焦成像和单离子通道活性的录音的示意表示孤立的啮齿类动物肾小球。简要地说，将大鼠麻醉后，肾脏应用PBS冲洗以清除血液它们。然后，将肾脏切除和解封装，并且肾小球从肾皮质通过差筛分分离。样品的部分可采取膜片钳分析，其余的可以装载荧光钙染料的Fluo-4，AM和的Fura红，AM，以执行共焦比率钙成像。 单离子通道活性的电测量可以肾小球的分离后马上执行。离子查活动nnels所用的膜片钳方法的细胞贴附和全细胞构型进行测量两者。为了证明所示的方法的可行性是单TRPC状钙传导离子通道活性（ 图2）的记录。此外，也可以应用细胞渗透性或受体结合的药物，以测试其对在单离子通道的电平的钙内流中的足细胞的效果。 一种方法，它允许单个离子通道活性的评估产生的各种数据，可以表征足细胞功能。然而，它可以大大地通过测量在全细胞的水平中的钙浓度变化的补充。为了执行这样的研究中，新鲜分离的肾小球中填装的荧光染料用于高通量共焦成像。具有高的光学分辨率是可能的监测几个足一次钙的水平。 图3表明了时间当然对于典型的实验设计用于评价钙浓度的足细胞中的变化。基于所述的Fluo-4的荧光足细胞内基础钙浓度进行评价。记录所述基底的信号强度，持续1分钟（F）之后，离子霉素被施加并引起峰荧光（F 最大 ）来达成，其然后通过的MnCl 2猝灭（F min的说明）。根据公式6.3，将Fluo-4信号在实验的每个时间点的强度，可再翻译成在细胞中的钙离子的实际浓度。如从图中看到的，平均荧光背景（约400任意单位）转化为140纳米，这是一个正常的浓度范围为在健康非凋亡细胞内钙内。 所描述的方法的重要性和实用性被其他应用程序，它允许测量急性钙transie合理在响应于各种药物的足细胞的nts。 图4A示出了大鼠肾小球在2mM的含钙溶液（在4.5中描述）染色的Fluo-4和的Fura红之前和加入10微米的ATP后的代表图像。请注意，在绿色的显着增加（的Fluo-4，AM）的足细胞的荧光（用箭头标记），并且在的Fura红荧光一滴（红色伪）。 Fluo4和FuraRed反映在相反的方式，即 488纳米的激发波长的增加钙浓度，当在细胞内钙离子浓度增加的Fluo 4强度上升，而FuraRed下降。这是可能由于FuraRed荧光团的双激发性质。根据激发波长可以表现得像钙结合（420纳米）或无钙（488纳米）的荧光团与荧光相应增加或减少。因此，单独在一个比率成像模式下的使用FuraRed的是可能的，但是它requir上课两个激励波长 - 420纳米和488纳米。这将至少两次（相比，使用一个激励波长的）减小成像频率;如果研究人员想要保持影像的快节奏，图像分辨率应减少。然而，将Fluo 4和FuraRed可以一起使用以比例方式而不分辨率或减少在成像频率损失，因为这些荧光团可以用相同的488纳米激光激发，并提供了发射光谱的良好分化。一个典型的短暂总结从感兴趣区标有在图4A的箭头示于图4B;该图清楚地表明加入10μMATP，在几分钟之内其衰减后的急性增加Fluo4 / FuraRed强度比。拍摄图像每隔4秒，因此，该技术允许观察在细胞内的钙浓度的快速变化。 图实验方案1.示意图 。后的动物被适当麻醉并为手术准备，​​肾脏被刷新用PBS以除去血液。然后，将肾脏切除和解封装，并且肾小球从肾皮质通过差筛分分离。在这里，部分样品被采取膜片钳分析，其余装有荧光钙染料和执行比例共聚焦钙成像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52850/52850fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52850/52850fig2highres.jpg" width="700" /> 图2.代表记录表示在PODO的TRPC钙通道的活性新鲜分离的大鼠肾小球核细胞。左图演示了一个电生理记录在细胞连接的配置中连接到肾小球的表面上的足细胞体的膜片钳吸管;一个封装肾小球的一部分可以看出，在图像的右下角。从在-40毫伏保持电位作出对大鼠肾小球的足细胞的细胞贴附膜片的TRPC状通道活性的代表性的电流曲线示出在右边。 C和O I表示封闭式和开放式通道的水平，分别为。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52850/52850fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52850/52850fig3highres.jpg" width="500" /> 图3.代表性的实验目的是评估在细胞内钙水平<html>足细胞。为了测量细胞内钙浓度，肾小球中装入的Fluo-4，AM，荧光强度被记录在基线和加法离子霉素和的MnCl 2的后。该图演示了荧光信号的变化响应于离子霉素（制造的Fluo-4的荧光，F 最大的最大值）和的MnCl 2，淬灭染料和结果的最低荧光强度（F 分钟 ）。荧光（左横坐标）对每个时间点的强度可以被翻译成实际的钙离子浓度在纳摩尔（右横坐标）根据下式在6.3。注意用足显示在时间点F，F 最大和F 分钟进行计算时，图像的边框。这里显示的图表反映了标有圆圈（ROI）足细胞的荧光强度;图像拍摄每4秒。NK"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/52850/52850fig4highres.jpg" width="650" /> 图响应ATP 4.测量在足细胞钙瞬变的 。 （ 一 ）代表图像说明野生型鼠肾小球前，加入10μMATP后的2毫米含钙溶液染色，荧光- 4（绿色伪）和红色的Fura（红色伪）。应用本药前低强度绿色的荧光应注意。 （ 二）总结例如细胞内钙瞬变通过应用10微米ATP诱发足的。注意在将Fluo-4 /的Fura红荧光强度比强烈而快速增加以下另外的药物，其占钙贮库和钙流入从第的刺激和耗尽的ê以外的细胞，从而导致细胞内钙浓度的升高。误差棒代表的手段的标准误差计算11足相同肾小球。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52850/52850fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

单通道分析和钙成像在新鲜分离的肾小球足细胞

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Nanyang Technological University

Research

JoVE Journal

Biology

10.9K 次观看.  Medical College of Wisconsin. Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli. 

视频: Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter&#39;s role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

科研

生物化学

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

神经科学

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

医学

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

生物学

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 ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

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 ...

<meta charset="utf8"></head><body>用于通道活化研究的单细胞钙成像

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

<meta charset="utf8"></head><body>用于研究钙离子通道激活的单细胞钙成像

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

用于研究钙离子通道激活的单细胞钙成像

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

单通道分析和钙成像在新鲜分离的肾小球足细胞

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使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像

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此视频中的章节

使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像

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