这项工作由美国国立卫生研究院GM62999，NIH EB007268，NIH AG15820，B＆B种子授予和NIH补充赠款给FR，BB奖学金，CM，CDT奖学金资助给RG。

幻灯片准备<ol><li>将22 mm x 40 mm玻璃显微镜放置在6 cm细胞培养皿中，每道1张。 </li><li>将每个载玻片的每一面以及6厘米的盘暴露于紫外（UV）光15-20分钟，以在无菌罩中消毒。 </li><li>用碟形玻璃盖上碟子，并放置在4°C直到准备使用。 </li></ol> 2.制备介质，缓冲液，溶液和试剂<ol><li>在去离子水中制备1升Hank&#39;s平衡盐溶液（HBSS），并加入10mM HEPES，5mM NaHCO 3和1mM EGTA。用NaOH调节至pH 7.2-7.3。将过滤缓冲液用0.22μm过滤器压入高压灭菌瓶中。 </li><li>在去离子水中制备900 mL高葡萄糖Dulbecco&#39;s Modified Eagle&#39;s Medium（DMEM）。加入44 mM NaHCO 3 ，并用HCl调节pH至7.2-7.3。将过滤介质用0.22μm过滤器压入高压灭菌瓶中。加入100 mL胎牛新鲜血清（FBS）对培养基使FBS浓度达到10％。储存于4°C。 </li><li>在去离子水中制备1μL细胞内缓冲液（125mM KCl，25mM NaCl，10mM HEPES，0.2mM CaCl 2，0.5mM EGTA，0.2mM MgCl 2 ），并将pH调节至7.25。最终的[Ca 2+ ]将为〜100nM。使用前，加入0.5 mM ATP。在室温下储存。 </li><li>在去离子水中制备1μL的林格缓冲液（121mM NaCl，2.4mM K 2 HPO 4，0.4mM KH 2 PO 4，10mM HEPES和1.2mM MgCl 2 ），并将pH调节至7.2。在室温下储存。使用，分批50mL 50mL管，并加入1.8 mM Ca 2+和10 mM葡萄糖。 </li><li>在去离子水中制备1L KCl漂洗溶液（125mM KCl，25mM NaCl，10mM HEPES，0.2mM MgCl 2 ），并将pH调节至7.25。 </li><li>将5mg的离子霉素溶于1mL二甲基亚砜（DMSO）中。将等分试样30μL加入微管并储存于-20℃6℃。 </li><li>通过将CPA溶解在DMSO中制备环戊酸（CPA）。确保添加到细胞中的CPA的最终百分比不超过1％，以防止细胞凋亡。通过溶解到过滤的H 2 O中并通过在37℃下振荡使其溶解过夜来制备20mM 4-氯 - 间甲酚（4-cmc）。通过将过滤的H 2 O溶解至100mM来制备ATP储备。 </li><li>为了确定原位 K d ，使用100μl的KCl缓冲液中的1mM EGTA，0.2mM，0.6mM，2mM，5mM，10mM，20mM，50mM和100mM Ca 2+ ， mM EGTA原液和溶解在去离子水中的1M CaCl 2原料。 </li></ol>细胞培养<ol><li>根据ATCC方案培养C2C12成肌细胞和HEK293细胞，每个在无菌UV罩中使用10cm细胞培养皿。 注意:C2C12细胞不允许生长高于约70％汇合，因为成肌细胞将开始分化进入肌管。 HEK293和C2C12细胞都容易生长，不应该允许生长超过100％融合以维持细胞完整性。另外，细胞在使用它们进行实验以保持细胞健康之前，不应该被传播10次以上。 </li></ol> 4. HEK293细胞的转染<ol><li>将种子HEK293细胞在6cm培养皿中的灭菌的22mm×40mm玻璃显微镜载玻片上，使其在转染当天〜70％汇合。 </li><li>第二天，按照制造商的方案转染试剂，使用2μgCatchER + cDNA和1:3（重量/体积）DNA:转染试剂比在还原血清培养基（ 如 Opti-MEM）中转染细胞。 </li><li>从盘中取出DMEM培养基，加入3 mL还原血清培养基。将DNA:转染试剂混合物加入到培养皿中，37℃孵育4-6小时。 </li><li>孵育后，用5-6mL HBSS洗涤细胞。舍弃HBSS f将盘取出，加入3mL新鲜的DMEM，37℃孵育48小时，以表达GECI。 </li><li> 转染C2C12成肌细胞 <ol><li>对于C2C12成肌细胞，通过添加足够的胰蛋白酶来覆盖盘底部（1-2mL），胰蛋白酶消化细胞。将培养皿置于37℃的培养箱中2-6分钟，使胰蛋白酶从培养皿底部松开细胞。 </li><li>一旦孵育完成，取出胰蛋白酶并将细胞吸入8mL的DMEM中。用DMEM彻底移取细胞，均匀分散并重新悬浮细胞，并将其种植在含有3mL DMEM的6cm细胞培养皿中的无菌盖玻片上，使最终汇合率达到60％。 </li><li>在同一天内转染细胞，如步骤4.2-4.3所述。在37°C孵育24小时。 </li><li>重复步骤4.4。 </li></ol></li></ol>幻灯片和荧光显微镜的准备<ol><li>打开m显微镜和光源，然后打开简单PCI程序。允许显微镜预热15-20分钟，或直到电荷耦合器件（CCD）相机冷却至-65°C。准备室，等待时用细胞滑动。 </li><li>使用棉签将覆盖低密度开放金刚石浴成像室的底部用薄层密封剂覆盖。 </li><li>将含有转染细胞的载玻片从培养箱中取出，并用预先温育至37℃的10mM葡萄糖和1.8mM Ca 2+的 1mL Ringer缓冲液轻轻冲洗三次。 </li><li>将1mL的林格缓冲液放在盘中，以防止细胞干燥，并使用镊子将滑块从盘中取出。通过将载玻片的边缘接触到实验室组织，使多余的溶液吸收到实验室组织上。然后将其放在包含润滑脂的腔室的侧面，细胞面朝上，使用螺丝刀将油脂和固定室固定在载物台上。 </li><li>添加1mL的林格缓冲液到室，以防止细胞干燥，同时将腔室安装在舞台上，并完成显微镜设置的其余部分。一旦真空抽吸软管连接到腔室，腔室容纳的最终体积为〜450μL。 </li><li>将显微镜物镜切换到40X油浸物镜。 </li><li>使用镜头纸和镜头清洁液清洁和干燥物镜。不要擦目的。轻轻擦拭直至表面清洁。 </li><li>将一滴浸油加入目标。 </li><li>将支架放置在显微镜平台上，并将真空尖端添加到腔室。一旦真空抽吸软管连接到腔室并打开，腔室容纳的最终体积为〜450μL。该最终体积与腔室的侧面平齐。在实验过程中，室解决方案必须保持水平，以防止由于过度填充而使溶液溢出到物镜中。 </li><li&gt;提高目标，并使用粗调，直到物体上的油接触到幻灯片的底部。 </li><li>使用亮场模式，将增益调整为175，将曝光时间调整为0.03秒，以对焦单元格。 </li><li>一旦细胞聚焦，使用488 nm激发切换到荧光模式。将曝光时间更改为0.07秒。找到具有足够的细胞健康的足够的荧光图像的视野。 </li><li>一旦选择了视野，在荧光和明场模式下进行拍摄。 </li><li>在单元格中圈出感兴趣区域（ROI），或者将整个单元格圈出以记录所选区域的强度。 </li></ol> 6.成像药物诱导的Ca 2+瞬态和原位 K d校准<ol><li>启动强度测量，帧速率每5秒设置一次。 </li><li>允许基线强度稳定20-30帧（100-150秒）。如果需要，减少fr这个步骤中的ames / s，以防止漂白。 </li><li>基于最终室体积〜450μL计算20mM储备液所需的4-cmc的量，使终浓度为200μM。根据需要稀释稀释液。 </li><li>小心地从室中取出60μL的林格缓冲液，并置于微量离心管中。用该溶液稀释计算出的4-cmc的量，并加回到室中以引起来自被成像的细胞的预期响应。 </li><li>将计算出的4cmc的量添加到微量离心管中并通过移液混合。 </li><li>在视场中轻轻添加该解决方案，同时在强度测量中添加事件标记。 </li><li>让药物与受体结合的时间和随后的信号减少，然后用同时添加事件标记物的同时用含有1.8mM Ca 2+和10mM葡萄糖的3-6mL的林格缓冲液洗涤。由于室容积为450℃，＃181; L并连接到真空，加入3-6mL缓冲液确保含有药物的前者溶液已被冲洗掉并用新添加的溶液替代。 </li><li>对于100μMATP和15μMCPA重复6.1-6.7，使用每个测定的转染细胞的新载玻片。 </li><li>测量完成后，在简单PCI程序的强度测量窗口中结束数据采集。 </li><li>拍摄细胞的荧光和明亮照片。 </li><li>打开实验的数据文件。在这里，如果需要，重新圈出感兴趣的细胞或区域以重新计算强度值。 </li><li> 为了确定C2C12成肌细胞中的原位 K d ，按照4.2节和第5节所述的方案，并将1mL的0mM Ca 2+ Ringer缓冲液置于室中。 <ol><li>用0.002-0.005％皂角苷在细胞内缓冲液中使细胞渗透15-30秒，以清除任何细胞CatchER +可能在细胞溶质中。用含10μM离子霉素的KCl缓冲液中的3-6mL 1mL EGTA洗涤细胞。允许强度降低，直到达到平台。 </li><li>用3-6毫升具有10μM离子霉素的KCl缓冲液冲洗，并使强度稳定，然后加入步骤2.7中详述的每种Ca 2+溶液。允许强度在每次添加之间达到平台。 </li></ol></li><li>要校准基础[Ca 2+ ]定量的传感器，请按照6.12步骤进行。使用EGTA和来自步骤2.7的100mM Ca 2+缓冲液以获得最小和最大荧光强度。 </li></ol> 7.数据处理<ol><li>下载强度测量数据的电子表格文件。 </li><li>将每个单元格的数据归一化为基线强度（F / F 0 ）。在任何图形程序中绘制归一化数据与时间的关系。 </li><li>要计算％变化，减去归一化峰值ue从1乘以100.计算多个细胞成像时的平均值和标准偏差。 </li><li>为了计算信噪比SNR，打开图像文件保存在图像处理软件ImageJ中，然后测量信号，转染细胞和噪声背景。使用方程SNR =信号/噪声计算SNR。 </li><li>为了从收集的荧光成像数据中计算原位 K d ，在加入每种Ca 2+浓度和EGTA（0mM Ca 2+ ）之后平均包括平台的强度数据。使用θ=（F-F min ）/（F max -F min ）计算分数饱和度（相对强度），其中θ是相对强度，F是任何点的荧光，F min是最小荧光强度， F max是荧光强度的最大值，以观察t的强度变化他与最小强度相比，加入Ca 2+进行探测。在任何图形和曲线拟合程序中绘制相对强度数据[Ca 2+ ]。使用任何图形和曲线拟合程序翻译的1:1结合方程θ= [M T ] /（K d + [M T ]）来计算K d 。 M T是总金属浓度。 </li><li>为了从6.13中概述的校准中量化基础钙，在加入1mM EGTA（F min ）和100mM Ca 2+ （F max ）后平均含有平台的强度数据。使用校准方程[Ca 2+ ] = K d （[F - F min ] /（F max - F）]计算基础钙。 </li></ol>

<ol>
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作者没有什么可以披露的。

荧光探针如CatchER +的活单细胞成像是分析每个细胞响应于受体激动剂或拮抗剂的复杂ER / SR Ca 2+信号传导过程的有效技术。这种技术对于同时使用多个波长的成像也是有用的，例如Fura-2所需要的或将Catcher +和Rhod-2一起图像分别监测ER和胞质钙变化。该协议有几个关键步骤;细胞转染可以对成像的可行性有很大的影响。低转录率可能导致GECI表达不足以监测钙的反应。另一方面，CatchER +的过表达不会对ER / ER Ca 2+产生缓冲作用，这与合成钙染料有关。 Ca 2+探针的缓冲效应受细胞器中的[Ca 2+ ]，p的K d的影响长袍和测量所需的探头浓度。细胞溶质[Ca 2+ ]通常处于低纳摩尔范围内，然而，所需的探针浓度在相当的范围内。在这种情况下，染料浓度的量及其缓冲细胞溶质钙的能力是细胞成像的主要关注点。相比之下，我们为不同的哺乳动物细胞系确定了ER / SR [Ca 2+ ]在100摩尔至毫摩尔范围内31 。由于CatchER +的0.1-1μM表达足以使得能够检测到ER钙，所以CatchER的缓冲作用可以忽略不计。因此，CatchER +可以监测高[Ca 2+ ]环境中的Ca 2+通量，而对腔内ER / SR Ca 2+ 32没有任何缓冲作用。 几个因素可能影响GECI的转染效率，如孵化时间，the转染试剂，孵育温度和细胞融合。当细胞接种在载玻片上时，细胞融合可以显着影响成像质量。低汇合可导致细胞数量少（n）和较少的统计准确度，而高水平的融合导致细胞重叠层在成像中产生大变量。转染时间和温度应根据细胞类型进行优化。另外，添加减少血清培养基的DMEM对于更长的转染时间是最佳的，以防止细胞凋亡。哺乳动物细胞中原始的GECI CatchER荧光在培养和转染时仅为30℃。 CatchER的荧光成功优化，并在37℃下表达得到改善，从而得到了新的改进型CatchER + 。可以使用针对EGFP的抗体进行免疫印迹实验以确认Ca 2+探针的表达。 而且，我方法和试剂递送的一致性至关重要。可以通过灌注，小体积扩散或机械泵将试剂加入到腔室中，所有这些都可引起不同的反应。必须通过在将放置在载玻片上的腔室的底部上施加一层密封剂脂膏来适当地密封溶液室。必须为探头选择正确的波长。 CatchER +的激发和发射波长分别为395/488 nm和510 nm。对于细胞成像，仅使用488nm激发以避免UV光对细胞的不利影响。因此，使用在488nm激发并且在510nm处收集发射的任何光学滤光器将是可接受的以用于使用CatchER +进行成像。为了防止光漂白，可以集中力量来优化光强度和曝光，如帧/ s 33 。 虽然这个协议是理想的分析效果使用CatchER + Ca 2+ ER / SR荧光探针进行ER / SR变化，在任何实验中都有预期的限制。由于单细胞活体成像仅分析细胞的一小部分，平均而言，细胞数（n）可以从1-100个细胞变化，但对于较大的细胞系而言可以更低。这导致较低的细胞数量和统计学上变化的结果，没有多次试验。统计准确度需要n&gt; 6。要获得更大的n，许多菜肴必须成像，或者必须同时使用其他技术。此外，CatchER +是单波长ER / SR Ca 2+探针，这导致其他单波长探针和染料的问题，无法量化信号，不知道信号是否为真，而不是细胞的破坏与比率系统相比，噪声较大。 这项工作表明，这些优化的Ca 2+探针可以应用于不同的细胞类型或组织类型，以监测受体介导的ER / SR Ca 2+释放。 CatchER +是单波长ER / SR Ca 2+探针。因此，重要的是实验参数和设置对于定量测量是一致的。细胞系中钙浓度和钙传感器K d的详细校准是定量分析的另外重要测量。 这些开发的钙传感器也可以针对ER / SR内的特定位置，以监测与各种生物和病理状况下的全球Ca 2+变化相比存在的大量不同的Ca 2+瞬变。这些发现将继续推动Ca 2+成像和探针设计领域，为诊断与Ca 2+相关的疾病提供未来的工具。报告的方案和开发的传感器也可以适用于药物d针对与钙信号传导相关的疾病的临床。

细胞内钙（Ca 2+ ）瞬态的时空属性激活各种生物学功能1 。这些Ca 2+信号事件通过不同的刺激细胞外触发，并由主要的Ca 2+储存细胞器和许多Ca 2+泵，通道和Ca 2+结合蛋白在细胞内控制。由于信号调制的缺陷，Ca 2+瞬变可能会显着改变，导致不同的疾病2 。由于Ca 2+信号传导系统的速度和复杂性，与内质网（ER）和肌质网（SR）在中心，需要基因编码的Ca 2+探针已被优化用于具有快速动力学的哺乳动物表达观察不同细胞的全球和局部Ca 2+变化3 。 ER和SR，其对应的肌肉细胞，是主要的细胞内Ca 2+储存细胞器，并充当Ca 2+水槽，有助于放大Ca 2+信号4 。 ER / SR是Ca 2+信号的组成部分，具有作为信号5的发射器和接收器的双重作用。兰诺定受体（RyR）和肌醇1,4,5-三磷酸受体（IP 3 R）是位于由Ca 2+ 6调节的ER / SR膜上的Ca 2+释放受体。其他药物直接或间接刺激这些受体的功能。 4-氯间甲酚（4-cmc）是RyR的有效激动剂，其具有比用于诱导SR Ca 2+释放的咖啡因高10倍的灵敏度，其中两者均经常用于研究RyR介导的Ca 2+释放健康和患病细胞7 。 ATP增加IP 3介导的Ca 2+通过IP 3 R 8租赁。 ATP结合嘌呤能受体P2YR，一种G蛋白偶联受体（GPCR），触发产生IP 3 ，结合IP 3 R从ER 9,10释放Ca 2+ 。肌质内质网钙ATP酶（SERCA）泵是一种P型ATP酶泵，也位于ER / SR膜上，可减少细胞溶质Ca 2+ ，并通过主动将离子注入ER / SR内腔来补充ER / SR 11 。 SERCA泵的特异性抑制剂包括来自加拿大的Thapsia garganica的thapigargin和来自曲霉和青霉的环磷酸（CPA）。 CPA对泵具有低亲和力，并可逆地阻断Ca 2+接入点12 。另一方面，毒胡萝卜素在M3螺旋中的残留物F256与纳摩尔不可逆地结合到不含Ca 2+的泵ar亲和力11 。分析和量化涉及Ca 2+刺激事件的变化已经并且仍然是一个挑战。由于ER / SR是Ca 2+信号传播过程中具有中枢功能的主要亚细胞Ca 2+含量隔室，因此大部分工作集中在了解ER / SR Ca 2+信号5 。 合成的Ca 2+染料的创建有助于推动Ca 2+成像的领域和实践。尽管诸如Mag-Fura-2的染料已被广泛用于测量不同细胞中的间隔Ca 2+ ，但它们具有诸如染料负载不均匀，光漂白和不能针对特定细胞器的限制。绿色荧光蛋白（GFP）的发现和荧光蛋白 -基于Ca 2+的探针已经推动了Ca 2+成像领域的发展。一些现有的GECI是涉及黄色荧光蛋白（YFP），青色荧光蛋白（CFP），钙调蛋白和M13结合肽17,18的 Förster共振能量转移（FRET）对。肌钙蛋白C基GECI也可作为FRET对的CFP和柠檬酸和单一荧光团探针19,20,21 。 其他，如GCaMP2和R-GECO是涉及钙调素22,23的单一荧光团传感器。为了克服狭窄调节K d和与其Ca 2+结合结构域24中发现的多个Ca 2+结合位点相关的协同结合的限制，一类新的钙通过在增强型绿色荧光蛋白（EGFP） 25,26的发色团敏感位置设计β桶表面上的Ca 2+结合位点来产生细胞。这种称为CatchER的高度传播的传感器在扩散极限附近具有〜0.18mM的k d ，在700s -1附近ak。 CatchER已被用于监测不同哺乳动物细胞系（如HeLa，HEK293和C2C12 25）中受体介导的ER / SR钙释放。由于其快速动力学，CatchER用于年轻和老朋友病毒B NIH Jackson（FVB）小鼠的屈肌腱肌肉纤维（FDB）肌纤维，以揭示在FDB中经过2秒的去极化后，更多的Ca 2+保留在SR中与老鼠相比，老鼠的纤维27 。克服其在37℃的低荧光，这阻碍了其在哺乳动物的钙成像中的应用细胞，我们开发了一个名为CatchER +的改进版本的CatchER。 CatchER +在37℃下表现出增强的荧光，以更好地应用于哺乳动物细胞。在CatchER中加入了另外的突变以提高37℃ 28,29 ℃的热稳定性和荧光，以产生CatchER + 。 CatchER +与CatchER 30相比，其信噪比（SNR）提高了6倍。 在这里，介绍了使用CatchER +培养和转染HEK293和C2C12细胞的方案及其用于监测ER / SR受体介导的钙瞬变的方案。显示了在用4-cmc，CPA和ATP处理的HEK293细胞中表达的CatchER +的代表性结果。我们还提供了一种用于确定C2C12成肌细胞和qua中CatchER +的 原位 K d的方案基础[Ca 2+ ]的鉴定。

<table><tbody><tr><td>4-Chloro-3-methylphenol (4-CmC)</td><td>Sigma-Aldrich</td><td>C55402</td><td></td></tr><tr><td>515DCXR dichroic mirror</td><td>Chroma Technology Corp.</td><td>NC338059</td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A26209</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>EMD Millipore</td><td>102382</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes (100 mm)</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes (60 mm)</td><td>Sigma-Aldrich</td><td>CLS430166</td><td></td></tr><tr><td>Cyclopiazonic Acid (CPA)</td><td>EMD Millipore</td><td>239805</td><td></td></tr><tr><td>D(+)-Glucose</td><td>ACROS Organics</td><td>41095-0010</td><td></td></tr><tr><td>Dow Corning 111 Valve Lubricant &amp;amp; Sealant</td><td>Warner Instruments</td><td>64-0275</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D7777</td><td></td></tr><tr><td>Ethylenebis(oxyethylenenitrilo)&lt;br/&gt; tetraacetic Acid (EGTA)</td><td>ACROS Organics</td><td>409911000&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher</td><td>26140087</td><td></td></tr><tr><td>Fisherbrand Cover Glasses 22x40 mm</td><td>Fisher Scientific</td><td>&amp;nbsp;12-544B</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salts (HBSS)</td><td>Sigma-Aldrich</td><td>H4891</td><td></td></tr><tr><td>HEPES, Free Acid, Molecular Biology Grade</td><td>EMD Millipore</td><td>391340</td><td></td></tr><tr><td>Immersion Oil without autofluorescence</td><td>Leica</td><td>11513859</td><td></td></tr><tr><td>Ionomycin, Free Acid</td><td>Fisher Scientific</td><td>50-230-5804</td><td></td></tr><tr><td>Leica DM6100B inverted microscope with a cooled EM-CCD camera</td><td>Hamamatsu</td><td>C9100-13</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher</td><td>11668019</td><td></td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>ThermoFisher</td><td>L3000015</td><td></td></tr><tr><td>Low Profile Open Diamond Bath Imaging Chamber</td><td>Warner Instruments</td><td>RC-26GLP</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>Opti-MEM</td><td>ThermoFisher</td><td>51985034</td><td></td></tr><tr><td>Potassium Chloride</td><td>EMD Millipore</td><td>PX1405</td><td></td></tr><tr><td>Potassium Phosphate, Dibasic</td><td>EMD Millipore</td><td>PX1570</td><td></td></tr><tr><td>Potassium Phosphate, Monobasic</td><td>EMD Millipore</td><td>PX1565</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>SimplePCI Image Analysis Software</td><td>Hamamatsu</td><td>N/A</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher Scientific</td><td>S233-3</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Sterivex-GV 0.22 &amp;micro;m filter</td><td>EMD Millipore</td><td>SVGVB1010</td><td></td></tr><tr><td>Till Polychrome V Xenon lamp</td><td>Till Photonics</td><td>N/A</td><td></td></tr><tr><td>Trypsin (2.5%), no phenol red (10x)</td><td>ThermoFisher</td><td>15090046</td><td></td></tr></tbody></table>

monitoring er/sr calcium release with the targeted ca2+ sensor catcher+

细胞外刺激诱发的细胞内钙（Ca 2+ ）瞬态引发了生物体内的多种生物过程。在细胞内钙释放的中心是细胞内主要的钙储存细胞器，内质网（ER）和肌肉细胞中更专门的肌浆网（SR）。来自这些细胞器的钙的动态释放由兰诺定受体（RyR）和肌醇1,4,5-三磷酸受体（IP 3 R）介导，通过肌肉/内质网钙ATP酶（SERCA）泵进行再灌注。创建了一种称为CatchER的遗传编码钙传感器（GECI），用于监测ER / SR的钙释放速度。在这里，HEK293和C2C12细胞中改良的ER / SR靶向GECI CatchER +的转染和表达的详细方案及其在监测IP 3 R，RyR和SERCA泵介导的钙瞬变中的应用在HEK293细胞中使用荧光显微镜概述。受体激动剂或选择的抑制剂分散在室溶液中，并且强度变化被实时记录。使用这种方法，用4-氯 - 间甲酚（4-cmc）的RyR活化，用三磷酸腺苷（ATP）间接激活IP 3 R和抑制具有环磷酸腺苷酸的SERCA泵，观察到ER钙的降低酸（CPA）。我们还讨论了确定C2C12细胞中原位 K d和定量基础[Ca 2+ ]的方案。总之，与CatchER +结合使用的这些方案可以从ER中引发受体介导的钙释放，并将来应用于ER / SR钙相关病理学研究。

本节将说明使用先前描述的使用优化的ER / SR靶向GECI CatchER +的方法通过不同的受体介导途径监测ER / SR Ca 2+的变化而实现的结果。 图1说明通过用200μM4-cmc刺激的RyR排空ER。 4-cmc是RyR的激动剂。通过CatchER +荧光强度的降低测量，添加药物会诱导ER钙的降低。如标示，本文概述的方案允许使用CatchER +观察和测量受体介导的Ca 2+瞬变，其提供关于ER Ca 2+的变化的信息。 如图2所示 ，加入15μMCPA，可逆地阻塞SERCA泵，产生形成高原的荧光强度的大幅降低。由于SERCA泵的功能被抑制，ER Ca 2+释放通道仍然主动释放Ca 2+到细胞质中，导致荧光强度降低。用含有1.8mM Ca 2+和10mM葡萄糖的林格缓冲液洗涤CPA后细胞恢复。这些结果证实了CatchER +监测SERCA泵特异性Ca 2+瞬变的能力。 图3显示了用CatchER +转染的HEK293细胞中具有200μMATP的IP 3 R间接激活的代表性数据。 ATP结合P2Y受体，一种G蛋白偶联受体，其产生通过IP 3 R激活Ca 2+释放的IP 3。虽然变化小，加入200μMATP可以显着降低信号强度。用含有1.8mM Ca 2+和10mM葡萄糖的林格缓冲液洗涤细胞可使ER再填充并使信号返回到基线。这些结果突出了CatchER +通过间接刺激监测IP 3 R介导的钙释放的能力。 在图4中 ，将CatchER +转染入C2C12成肌细胞中以确定原位 K d 。按照方案中的说明收集数据。将C2C12成肌细胞用0.005％皂角苷透化15-30秒，然后用含有10μM离子霉素的KCl缓冲液中的1mM EGTA洗涤。在含有10μM 离子霉素的KCl缓冲液中制备各种Ca 2+浓度 ，并以逐步方式加入。将数据归一化并使用1:1结合进行拟合方程式。 7个细胞的平均K d为3.1±1.4mM。 CatchER +的弱亲和力允许它感知高Ca 2+细胞器中的Ca 2+，如ER或SR。为了确定SR中的基础[Ca 2+ ]，将C2C12细胞用0.005％皂角苷透化15-30秒，用KCl缓冲液洗涤，然后用含有10μM离子霉素的KCl缓冲液中的1mM EGTA洗涤以获得F min 。用KCl缓冲液洗涤EGTA后，加入最大值为10μM，最高为100μMCa 2+的 F max 。基础Ca 2+计算为0.4±0.2mM。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55822/55822fig1.jpg" /> 图1:在HEK293细胞中使用4-cmc刺激RyR。 （ A ）用4-cmc描绘RyR的激活。 （ B ）HEK293细胞的强度记录用CatchER +转染，定位在ER中，用200μM4-cmc处理。用4-cmc刺激RyR导致归一化荧光强度的降低与[Ca 2+ ] ER的降低直接相关。 n是指成像的细胞数。平均信号减少为12.0±2.2％。在林格氏缓冲液中存在1.8mM Ca 2+有助于将强度恢复反映的ER再填充到基线。图像显示用4-cmc处理前后的细胞。 SNR计算为4.3±0.8。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55822/55822fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55822/55822fig2.jpg" /> 图2:使用可逆SERCA p减少[Ca 2+ ] ERUP抑制剂CPA在HEK293细胞中。 （ A ）CPA对SERCA泵的抑制作用。 （ B ）用CatchER +转染的HEK293细胞的活体成像，定​​位在ER中，用15μMCPA处理。由于Ca 2+仍然可以通过IP 3 R和RyR流动，所以使用CPA阻断SERCA泵导致归一化荧光强度的降低，这与[Ca 2+ ] ER的减少直接相关。 n是指成像的细胞数。插图是在处理前后用CatchER +转染的HEK293细胞。平均信号下降21.0±0.3％。在林格氏缓冲液中存在1.8mM Ca 2+有助于将强度恢复反映的ER再填充到基线。 SNR计算为5.5±0.8。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55822/55822fig2large.jpg" target="_blank">请点击这里查看更大的版本</a>这个数字。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55822/55822fig3.jpg" /> 图3:HEK293细胞中ATP降低[Ca 2+ ] ER 。 （ A ）通过嘌呤能受体P2YR与ATP间接激活IP 3 R的示意图。 P2YR是在ATP结合时产生IP 3的GPCR。 IP 3激活IP 3 R，刺激ER释放钙。 （ B ）使用CatchER +转染的HEK293细胞进行实时成像，定位在ER中，用200μMATP处理。通过P2Y受体与ATP间接刺激IP 3 R导致与[Ca 2+ ] ER的降低直接相关的归一化荧光强度的降低。 n是指成像的细胞数。平均信号下降6.0±3.0％。 SNR为c计算为6.7±2.7。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55822/55822fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55822/55822fig4.jpg" /> 图4:C2C12成肌细胞中CatchER +的 原位 K d 。从用CatchER +转染的透化的C2C12成肌细胞细胞收集的归一化强度的代表性痕迹。细胞用细胞内缓冲液中的0.005％皂角苷透化15-30秒。在KCl缓冲液中制备EGTA和Ca 2+溶液，n表示成像的细胞数。 （ A ）插入荧光图像代表治疗前后的细胞。在10μM的存在下，向透化的C2C12成肌细胞中加入0.3,0.6,2,5,10,20,50和100mM Ca 2+离子霉素得到3.1±1.4mM的K d 。 （ B ）在分别加入1mM EGTA和100​​mM Ca 2+ ，分别加入10μM离子霉素后，Inset荧光图像代表最小和最大值的细胞。基础Ca 2+计算为0.4±0.2mM。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55822/55822fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+ at JoVE.com

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

我们提出了使用我们的靶向基因编码钙指示剂（GECI）CatchER +的方案 ，用于使用实时荧光显微镜监测HEK293和骨骼肌C2C12细胞的内质网/肌浆网（ER / SR）中的快速钙瞬变。还讨论了原位测量和校准的协议。

监测ER / SR钙释放与目标Ca<sup&gt; 2+</sup&gt;传感器捕捉器<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

monitoring-ersr-calcium-release-with-the-targeted-ca2-sensor-catcher

Nanyang Technological University

Research

JoVE Journal

Biology

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

14.8K 次观看.  Georgia State University. 我们提出了使用我们的靶向基因编码钙指示剂（GECI）CatchER +的方案 ，用于使用实时荧光显微镜监测HEK293和骨骼肌C2C12细胞的内质网/肌浆网（ER / SR）中的快速钙瞬变。还讨论了原位测量和校准的协议。 

视频: Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

监测变化的细胞内钙离子浓度和突触效能的软体动物<em&gt;海兔</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

科研

神经科学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

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, ...

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

肌网钙储备与细胞内舒张钙在心室肌细胞中的分离评价

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

医学

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

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, ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

监测ER / SR钙释放与目标Ca

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膜靶向 ca 2 + 指标对培养细胞中局部 Ca^{2 +}信号的解剖

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

成像本地CA

监测内质网钙稳态使用