SL是从东风集团（研究生研究学院845）的奖学金支持。这项工作是由东风集团（SFB的530/C1＆967）赠款，由HOMFOR支持。

1。股票方案的制备<ol><li>准备无钙缓冲液（140毫米氯化钠，氯化钾，5毫米1毫米氯化镁 2，0.5毫米EGTA，10毫米的葡萄糖，在10毫米的HEPES - KOH（pH值7.35） ）。 </li><li>溶解FURA - 2 DMSO获得1毫米的原液。混合使用一个漩涡，直至溶液是均匀的浅黄色。保护灯杯。稀释FURA - 2原液上午到1毫升贝科的改良Eagle培养基（DMEM）加载HeLa细胞含有10％小牛血清（FBS），1％青霉素/链霉素的终浓度为4微米。 </li><li>准备在蒸馏水（pH值7.0）12.5毫米嘌呤原液。在-20 ° C店等分稀12.5毫米，在无钙缓冲嘌呤股票的解决方案到500微米的嘌呤的最终浓度。 </li><li>毒胡萝卜素溶解在DMSO中获得1毫米原液。 </li><li>溶解1毫克ophiobolin一个在20μL氯仿，并随后与25μL二甲基亚砜混合。的离心管中保持打开状态，直到氯仿蒸发。因此，最终ophiobolin A的浓度是100毫米。在-20 ° C店等分在使用之前，无钙缓冲储备液稀释成终浓度为100μm。因此，最终DMSO为活细胞成像的浓度不超过0.1％。 </li><li>溶解在DMSO中三氟到10毫米，存储等分集中在-20 ° C。无钙缓冲储备液稀释成终浓度为10μm，使用前。因此最终的DMSO浓度为活细胞成像，像ophiobolin一个，不超过0.1％。 </li><li>在核糖核酸酶自由水溶解的siRNA（SEC61A1 SEC61A1 - UTR的siRNA，siRNA和对照siRNA）准备20微米的原液。混合使用一个旋涡，并通过眼睛观察的增溶。商店50μL分装于-20 ° C </li></ol> 2。在HeLa细胞中的基因沉默为了研究某种蛋白质的贡献ER的Ca 2 +外流，分别的基因有两个不同的siRNA（图 1）要有效地沉默。此外，要克服各自的野生型基因表达的沉默效果。通常情况下，我们使用对编码和非编码区域（UTR），分别感兴趣的基因，定向的siRNA。采用非编码区，定向的siRNA提供了一个便捷的方式为互补。 <ol><li>种子5.2 × 10 5 HeLa细胞（ATCC没有。CCL - 2）在一个6厘米的培养皿中，用25毫米的盖玻片（聚- L -赖氨酸（1毫克/毫升）与200μL1小时前处理）在饱和湿度的环境中， 用 5％的CO 2（终体积为3.9毫升）贝科的改良Eagle培养基（DMEM）中含有10％胎牛血清（FBS）和1％青霉素/链霉素和孵化在37 ° C。 </li><li> SEC61A1 SEC61A1 - UTR的siRNA，siRNA或阴性对照siRNA的，使用HiPerFect的试剂，根据制造商的说明（终浓度的siRNA：20纳米）转染的细胞。简单地说，准备siRNAs的新鲜实际转程序之前，在一个单独的离心管：添加20μLHiPerFect转染试剂4μL（20μM）每个siRNA的是溶解在80μLOptiMEM。这种混合是轻轻振荡孵育，并在室温为10分钟。添加滴加种子5.2 × 10 5 HeLa细胞（3.9毫升）的siRNA结构（104μL）。 </li><li> 24 h后，改变培养基（3.9毫升）和转染的细胞，与第二次相同的siRNA组合（104μL）。 </li><li> Western blot分析评估沉默。它应在80％以上。洗细胞从细胞培养皿中的磷酸盐缓冲液（PBS）和收获的DMEM。通过采用伯爵夫人自动细胞计数器计数细胞。裂解细胞，在细胞裂解液（氯化钠10毫米，10毫米的Tris - HCl，pH值8.0，3毫米氯化镁 2，0,5％NP40）辅以一种蛋白酶抑制剂的鸡尾酒（3微克/毫升胃酶抑素，3微克/毫升3微克/毫升Antipain，亮肽素，3微克/毫升Chymastatin）和混合SDS样品缓冲液（60毫米的Tris - HCl，pH值6.8，2％SDS，10％甘油，5％β-巯基乙醇，0.01％溴酚蓝），获得了10.000细胞/μL终浓度。热样品在56 ° C下15分钟，随后，添加一些GLAS珠和20分钟的旋涡DNA片段。独立的20μL，每个样品由Laemmli型的SDS - PAGE（通常为15％聚丙烯酰胺分离胶）。 electroblotting（“湿污点”）在恒定的400 mA的3小时或过夜（100毫米，12.5毫米的Tris - HCl甘氨酸）在传输缓冲区转移到PVDF膜分离蛋白。随后，与5％（W / V）低脂肪奶粉的PBS在室温为30分钟，然后块暴露的印迹对C -末端的Sec61α兔执导的主要抗体，抗β-肌动蛋白抗体从鼠标。洗净的印迹（0.05％），PBS，TritonX - 100和TWICE的PBS分别为5分钟，。可视化的主要抗体，使用ECL Plex的山羊抗兔IgG抗体- Cy5的或ECL Plex的羊抗鼠IgG抗体，Cy3标记共轭和台风三星成像系统与图像定量TL软件7.0相结合的。 SEC61A1基因的情况下，最大的沉默效果通常是96小时后看到的第一个转。一个代表性的实验是在图所示。 2。 </li></ol> 3。 HeLa细胞的互补为了拯救SEC61A1沉默的表型 ，SEC61A1 cDNA插入到多克隆位点（MCS）的一个载体pcDNA3内部核糖体进入位点（IRES）- GFP的载体，含有巨细胞病毒（CMV）启动子的MCS，浮粉，加上绿色fluoresecent蛋白（GFP）的编码序列。 <ol><li>交换的媒介，第二次（3.9毫升）和改造，要么空载体或使用Fugene高清根据制造商的协议（矢量最终比例Fugene高清是4 微克 SEC61A1表达质粒的细胞48 h后的第一个siRNA的转载体，以16μLFugene HD）。简而言之，准备质粒刚在一个单独的离心管之前的转型过程：添加16μLFugene HD改造试剂每个质粒溶解在80μLOptiMEM到4微克。轻轻涡旋混合，在室温下孵育10分钟。加入质粒组合（0.1毫升）滴加种子5.2 × 10 5 HeLa细胞（3.9毫升）。 </li><li>经过48小时的表达质粒，荧光显微镜钙成像和收获前的主题培养皿。如果有必要更好的荧光信号的DMEM培养液以PBS代替。荧光显微镜配备冷却CCD相机记录荧光图像。转化效率可在明模式计算，并应在80％以上的细胞显示绿色荧光的细胞数量除以决定。一个典型的结果如图。 3。 </li></ol> 4。活细胞钙成像<ol><li>在测量之前，转让与转染的HeLa细胞涂在一个单独的3.5厘米的培养皿中的盖玻片。装入4微米FURA - 2在1毫升的DMEM，45分钟在25 °彗星在黑暗 11,12的细胞。 </li><li>修正了一个25毫米盖玻片iMIC金属腔和洗细胞两次，并在一个无钙缓冲液（每次300μL）孵育。 </li><li>彩瓷V iMIC显微镜在340 nm和380 nm交替兴奋开始收集数据，并发出荧光测量在510 nm（过滤器设置：分光镜，565 nm的发射器，纳米七十〇分之六百〇五）。样品含有20-50细胞/帧的图像，每3秒记录FURA - 2信号的比值F340/F380，F340和F380分别在340和380 nm，对应减去背景荧光强度。 </li><li> 1分钟后，在无钙缓冲或相同的缓冲处理的细胞嘌呤霉素（500微米）。另外，对待一个小分子抑制剂（如10μM三氟或100微米ophibolin一个）或细胞与无钙缓冲。 </li><li>比例测量后进行第2或10分钟，加毒胡萝卜素（1μM），并继续测量。 </li><li>最终，cytoslic的[Ca 2 +]是一个既定的标定方法比测量估计。Excel 2007和原产6.1分析数据。 </li></ol> 5。代表性的成果： 到目前为止，我们讨论问题（SEC61A1基因沉默是否影响钙的Ca 2 +）ER（图1）泄漏。 SEC61A1基因是由两个不同的siRNA在HeLa细胞96小时的沉默。虽然沉默几乎没有影响细胞的生长和活力，蛋白质的半通透细胞的ER的交通几乎完全被抑制。此外，SEC61A1沉默细胞受到严重影响钙2 +从ER泄漏。逆转的SEC61A1基因表达，基因沉默的效果。因此，Sec61复合物，在所有有核细胞的ER膜形成的Ca 2 +，可以预期，发挥了至关重要的作用在Ca 2 +稳态泄漏渠道。然而，大，装满水的毛孔不受控制的离子通透性Sec61在ER膜的复合物的形成，存在，将严重干扰与监管释放的 Ca 2 +从ER的管腔进入细胞质，细胞内的重要机制信号。我们确定了一个钙调蛋白（CAM）结合基序，在哺乳动物Sec61α胞质的N - 末端约束CAM 的 Ca 2 +纳摩尔亲和力和序列特异性apocalmodulin。在细胞水平上，两个不同的CaM拮抗剂钙刺激<sup&gt; 2 +从ER的存在，但不是在Sec61通道（图4和5）的情况下释放。后者没有观察时Sec61渠道，载于Sec61α智商图案的突变。因此，参与的Ca 2 + - CAM是限制的Ca 2 +从ER（ 图6）泄漏。 <img src="/files/ftp_upload/2730/2730fig1.jpg" alt="图1"/> 图1。流程图。有关详细信息，请参阅文本。 <img src="/files/ftp_upload/2730/2730fig2.jpg" alt="图2"/> 图2：沉默效率的数据分析。沉默了评价免疫印迹使用，对Sec61α和β-肌动蛋白（负载控制）的抗体分析。主要抗体，通过ECL Plex的二次抗体和荧光成像的可视化。 <img src="/files/ftp_upload/2730/2730fig3.jpg" alt="图3"/> 图3数据分析转染效率。配备冷却CCD相机荧光显微镜图像记录。转化效率可在明模式计数细胞显示绿色荧光的细胞数量除以决定。 <img src="/files/ftp_upload/2730/2730fig4.jpg" alt="图4"/> 图4中的屏幕快照，从活细胞成像HeLa细胞中的钙存在控制或SEC61A1 siRNA和CAM -拮抗剂ophiobolin A. HeLa细胞的存在或缺乏对SEC61A1（B）或一个执导的siRNA治疗阴性对照siRNA（A）为96小时表示。装有这些细胞的钙指标FURA - 2上午在CA 2 +孵育自由缓冲区缓冲液中含有0.5毫米EGTA，缓冲区或ophiobolin（蛇绿岩）和恢复的孵化。 10分钟后的Ca 2 +释放发起毒胡萝卜素（TG）申请在没有外部CA 2 +和继续孵化。在指定的时间从连续钙成像的屏幕截图。 <img src="/files/ftp_upload/2730/2730fig5.jpg" alt="图5"/> 图5数据分析活细胞钙离子成像实验。 （一）动能和定量分析图所示的一系列实验 。 4A。 Cytoslic的[Ca 2 +]比测量估计既定的标定方法2。毒胡萝卜素的效果显示为条形图。 （BD）的细胞与对照siRNA或48小时表示SEC61A1 - siRNA的治疗，要么控制向量（C） 或 SEC61A1表达质粒（C）或突变 SEC61A1表达质粒（四）所示，然后转化。 48 h后，钙离子成像实验进行了在图4和 5A的电流。胞质钙变化的统计分析 +此外，如在一个显示的实验后的毒胡萝卜素浓度。 P值&lt;0.001定义为显着的配对t检验，并表示由三个星号(***), NS，不显着。分析细胞的数字表示。平均值，误差棒代表的手段的标准误差（SEM）。我们注意到，这些例子改编自文献。 13。 <img src="/files/ftp_upload/2730/2730fig6.jpg" alt="图6"/> 图6：这些数据表明，新生链释放Sec61复杂，确实导致从ER和形成Sec61复杂的胞浆内口周围的钙nanodomain的钙离子释放。钙势必钙调蛋白和钙 - 钙调蛋白关闭Sec61复杂。“

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC5+is+a+Ca2+-activated+channel+functionally+coupled+to+Ca2+-selective+ion+channels.">TRPC5 is a Ca2+-activated channel functionally coupled to Ca2+-selective ion channels.</a> J. Biol. Chem. 284, 34423-34432 (2009).</li><li>Erdmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+calmodulin+with+Sec61a+limit+Ca2++leakage+from+the+endoplasmic+reticulum.">Interaction of calmodulin with Sec61a limit Ca2+ leakage from the endoplasmic reticulum.</a> EMBO J. 30, 17-31 (2011).</li></ol>

在哺乳动物细胞内质网（ER）中起着关键作用在蛋白质生物合成以及钙信号。在这里，我们描述了一个实验系统，使我们能够直接解决一个潜在的Ca 2 +泄漏通道的作用，并表征其公认的13个监管机制。这个实验系统为我们提供了独特的机会，以便i）评估在各种细胞类型，II的ER，不同的ER膜蛋白的贡献 ，从被动的Ca 2 +外流）特征的蛋白质和机制，限制了这种被动的Ca 2 +外流，并iii）研究疾病联系在一起的相关组件中的突变的影响。 我们注意到，唯一可行的细胞进行分析，并应在80％以上，整体可行性。因此，细胞活力，定期评估采用核ID蓝色/绿色的细胞活力试剂，根据制造商的协议。此外，在胞浆钙的变化进行统计分析+浓度是强制性的。因此，实验要进行四个不同批次的细胞和两个盖玻片必须为每个在一个单一的实验条件分析至少有20个细胞。我们注意到，在盖玻片后播种的同时，要进行各种实验。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> DMEM培养液+ GlutaMAX </td><td> Invitrogen公司</td><td> 31966 </td></tr><tr><td> OptiMEM + GlutaMAX </td><td> Invitrogen公司</td><td> 51985 </td></tr><tr><td>胎牛血清</td><td> Biochrom </td><td> S0115 </td></tr><tr><td>青霉素/链霉素</td><td>临时机场管理局</td><td> P11 - 010 </td></tr><tr><td> HiPerFect </td><td> Qiagen公司</td><td> 301707 </td></tr><tr><td> Fugene高清</td><td>罗氏诊断</td><td> 04709713 </td></tr><tr><td>核ID蓝色/绿色细胞活力试剂</td><td>恩佐生命科学</td><td> ENZ - 53004 </td></tr><tr><td> FURA - 2点</td><td> Invitrogen公司</td><td> F - 1221 </td></tr><tr><td>嘌呤霉素</td><td>西格玛</td><td> P 7255 </td></tr><tr><td>毒胡萝卜素</td><td> Invitrogen公司</td><td> T - 7459 </td></tr><tr><td> Ophiobolin一个</td><td>恩佐生命科学</td><td> ALX - 270 - 109 </td></tr><tr><td>三氟拉嗪</td><td>西格玛</td><td> T 6062 </td></tr><tr><td>伯爵夫人®自动细胞计数器</td><td> Invitrogen公司</td><td></td></tr><tr><td>台风三重奏成像系统</td><td> GE医疗集团</td><td></td></tr><tr><td> TE2000 - S的显微镜DS - 5Mc相机</td><td>尼康</td><td></td></tr><tr><td>彩瓷V iMIC显微镜</td><td>直到光子</td><td></td></tr></tbody></table>

live cell calcium imaging combined with sirna mediated gene silencing identifies ca2+ leak channels in the er membrane and their regulatory mechanisms

在哺乳动物细胞内质网（ER），起着关键作用在蛋白质生物合成以及钙信号 1 。在ER膜的异三聚体Sec61复杂提供了一个新进入管腔内质网合成的多肽水溶液的路径。最近从不同的实验室的工作表明，这种异源复杂，也可能形成瞬变的Ca 2 +泄漏通道 2-8 。重点观察这个概念是从核糖体和Sec61复杂的嘌呤导致瞬态释放的Ca 2 +从内质网，新生多肽的释放。此外，它已在体外观察，二管腔蛋白BIP是防止Sec61 复杂 9,10水平的离子通透性。我们已经建立了一个实验系统，使我们能够直接解决Sec61复杂的潜在的Ca 2 +泄漏通道的作用，并描述其公认的监管机制 11-13 。该系统结合的siRNA介导的基因沉默和活细胞的Ca 2 +影像13。细胞被视为是对编码和非编码区（UTR），分别在SEC61A1基因或负对照siRNA定向的siRNA 。互补分析，共同的细胞转染与IRES - GFP的抗性的野生型SEC61A1基因的siRNA表达载体，让。然后装入细胞与比例的Ca 2 +指标FURA - 2监测同时改变胞浆的Ca 2 +通过荧光显微镜观察细胞的数量，浓度。胞浆钙的连续测量 +还允许的各种药物的影响，如嘌呤，小分子抑制剂，毒胡萝卜素， 对Ca 2 +漏评价。这个实验系统为我们提供了独特的机会，以便i）评估在各种细胞类型，II的ER，不同的ER膜蛋白的贡献 ，从被动的Ca 2 +外流）特征的蛋白质和机制，限制了这种被动的Ca 2 +外流，并iii）研究疾病联系在一起的相关组件中的突变的影响。

Watch this Scientific Journal Video about Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms at JoVE

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

内质网在蛋白质生物合成和钙稳态的关键作用。我们已经建立了一个实验系统，使我们能够解决的Ca2 +泄漏通道的作用，并表征其公认的监管机制。该系统涉及的siRNA介导的基因沉默和活细胞内Ca2 +成像。

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 ...

live-cell-calcium-imaging-combined-with-sirna-mediated-gene-silencing

Research

JoVE Journal

Biology

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

18.0K 次观看.  Saarland University. 内质网在蛋白质生物合成和钙稳态的关键作用。我们已经建立了一个实验系统，使我们能够解决的Ca2 +泄漏通道的作用，并表征其公认的监管机制。该系统涉及的siRNA介导的基因沉默和活细胞内Ca2 +成像。

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

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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 ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

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

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

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

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

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 ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.