达尼洛Maddalo是作为年轻的调查组，从卡尔斯鲁厄理工学院（YIG）的研究奖学金获得者。

1。播种细胞<ol><li>开始前，热身在37℃水浴中培养液，PBS 1X和胰蛋白酶˚彗星</li><li>的孵化器中取出细胞，吸中等。 </li><li>轻轻地适用于洗细胞5毫升的PBS 1X。 </li><li>吸出PBS 1X和适用于1毫升胰蛋白酶含0025％EDTA。 </li><li>轻轻旋转板有均匀分布的胰蛋白酶。 </li><li>在孵化器中放置的细胞在37˚C为5-10分钟（取决于细胞类型）。从不定期检查，如果细胞分离摇板。 </li><li>在10-20毫升培养基重悬细胞，并收集他们在50毫升猎鹰管。 </li><li>使用贝克曼计数器计数细胞。细胞的数量应足以确保约60-70％汇合。 </li><li>板细胞，并允许为6-8小时，他们的种子。 </li></ol> 2。横贯染<ol><li>转染前，热身无血清培养基分装在37˚C。水浴中</li><li>带来的X极端基因9至室温。 </li><li>吸取无血清培养在2个Eppendorf管（1管= 1转）。 </li><li>吸管的X极端基因的无血清培养的重视，针尖不触及5分钟Eppendorf公司和孵化的塑料墙。在此期间准备两个DNA混合：一管混合在一起海肾，萤火虫和一个空的矢量控制的质粒，在另一个管海肾，萤火虫和分子伴侣。 </li><li>含无血清培养+ X极端基因9，旋涡和室温孵育20分钟到每个Eppendorf公司的DNA结构。 </li><li>滴加每个组合在37到24小时，每块板和孵化° C和5％的CO 2 </li></ol> 3。分裂的细胞<ol><li> 24小时后，转染，trypsinize如1.4中所述的细胞 - 1.6和稀释他们的60-70％，在6孔板最终汇合。 </li><li>准备一个盘子非震惊控制，并尽可能多的恢复时间点，要分析板， 显示图 。 2。 </li></ol> 4。热休克<ol><li>热休克，预温的含药血清在37℃水浴中语言˚C。 </li><li>准备“热休克缓冲区”，在含血清培养基酮稀释到终浓度为20微克/毫升和拖把至20毫米。 </li><li>现在拿出从孵化器板和吸出培养基。 </li><li>每孔1毫升的“热休克缓冲区，30分钟在37˚C和5％的CO 2（也包括在这种治疗方法的参考板） 。这将停止从头蛋白质的合成。 </li><li>在水浴上同时开关，并设置了T在45˚C。emperature </li><li>孵育后，密封封口膜板的盖子，以前在条纹削减。该参考板（100％refolfed萤火虫）是留在孵化器。 </li><li>在45˚C下30分钟的孵育板。 </li><li>从水浴中取出的钢板，删除的封口膜，并把板块的孵化器，允许恢复。 </li><li>在每个时间点收获细胞，并在PBS 1X洗4 1分钟˚C。离心在2000Xg </li></ol> 5。细胞裂解和荧光素酶检测<ol><li>对于一个高效的细胞裂解，单元冻结在液氮中的细胞沉淀。 </li><li>加入200μL双蒸水稀释1:5玻璃管被动裂解缓冲。 </li><li>在96孔板中加入30μL细胞裂解液。每个样品一式三份吸管。 </li><li> “非震惊”参考样本将被用来读海肾荧光素酶的活动，因为这一步是用来检查相等的转染效率。 </li><li>准备检测的解决方案。有关荧光素的解决方案稀释荧光素的终浓度为0.2毫米，在甘氨酸 - 甘氨酸缓冲。反应缓冲液稀释到1mm甘氨酸 - 甘氨酸缓冲终浓度DTT和ATP。腔肠反应缓冲液，稀释到终浓度为0.2μM腔肠缓冲腔肠。保持荧光素的解决方案和腔肠反应缓冲液避光。 </li><li>现在放置在光度计96孔板和启动程序“Wallac 1420工作站”。 </li><li>引入猎鹰含腔肠的解决方案管Pump1。盖上盖子，所以没有光可以管接触。 </li><li>选择从菜单中选择“饮水机维修”，并勾选Pump1。 </li><li>选择选项“补”。 </li><li>要编辑的板块布局，选择选项“探索协议和成果”菜单上的。选择协议，并双击就可以了。现在选择要读取的井。 </li><li>回到“Wallac经理”，然后单击“开始”。之间的海肾荧光素酶及其底物腔肠的反应发出的峰值波长为482纳米的光。 </li><li>阅读后，“饮水机维修”，选择“空”和Pump1管释放所有剩余的解决方案。 </li><li>现在，移动猎鹰管中水的管，并勾选“刷新”。 </li><li>冲洗步骤后，空管，通过选择“空”。 </li><li>放置在反应缓冲液猎鹰管从Pump1和荧光素猎鹰管Pump2管。 </li><li>更改板块布局。 </li><li>选择“补”Pump1和Pump2从“迪斯宾塞维护“菜单。 </li><li>选择协议，并开始阅读。之间的萤火虫荧光素酶及其底物荧光素的反应发出的峰值波长560纳米的光。 </li><li>在阅读结束重复上述步骤Pump1和Pump2空冲洗空。 </li><li>结果，现已在“Wallac 1420资源管理器”。 </li><li>每个实验相关的检测数量，用户名，开始和结束日期。 </li><li>结果现在可以导出为Excel文件。 </li><li>计算，细胞分为三组：1。非震惊的参考样本（设置为100％的复性萤火虫荧光素酶）; 2。无细胞分子伴侣和震惊热; 3。细胞与分子伴侣和热震惊。 </li></ol> 6。代表性的成果蛋白复性是直接相关的恢复，因此，测试的时间，如果系统正亲<html> perly，测定在不同时间点收集细胞进行。复性时间/百分比有着直接的关系表明，实验已正确执行，因此代表了一个限制因素。然而，应当始终认为热休克后的复性是饱和的事件，因此任何实验开始前，应执行适当的时候当然分析。 <img src="/files/ftp_upload/3540/3540fig1.jpg" alt="图1"/> 图1实验概述 。在体内复性实验需要3到4天完成。天1细胞接种和转染。翌日细胞劈裂到一个较小的格式，并在第3天，他们热震惊。在这一点上可能执行后收获细胞的荧光素酶检测或保存于-80 ° C细胞沉淀，并执行在另一时刻的检测_content“&gt; <img src="/files/ftp_upload/3540/3540fig2.jpg" alt="图2"/> 图2细胞分裂 。第2天细胞分裂成6孔板。每个转染将被摊薄，以及为了所有的同一个盘子转染。每个板块将对应一个特定的恢复时间，加上非震惊的参考板。 <img src="/files/ftp_upload/3540/3540fig3.jpg" alt="图3"/> 图3。代表好的结果 。 A.复性的百分比直接测定萤火虫荧光素酶的活性。每个条形图表示复性的情况下（黑网吧）和存在的分子伴侣在不同的恢复时间点（红色条）的百分比。 B.相关的情况下（黑线）和存在的伴侣（红线）之间的时间和复性。 R平方值显示良好的相关性。 在连接g.3A是一个细胞的情况下热休克（黑网吧），15，30，45，60和120分钟后存在的分子伴侣（红色条）收集的有代表性的成果。复性与长时间的恢复时间，并与分子伴侣的转染荧光素酶增加的百分比。显示控制（ 图3B，黑线）和转染细胞的分子伴侣（ 图3B，红线）的复性和时间之间的相关性。 <img src="/files/ftp_upload/3540/3540fig4.jpg" alt="图4"/> 图4。代表坏的结果 。 A.复性的百分比直接测定荧光素酶活性。每个条形图表示复性的情况下（黑网吧）和存在的分子伴侣在不同的恢复时间点（红色条）的百分比。 B.相关性的情况下，在存在时间和复性之间（红升INE）的伴侣。 R平方值表示坏的相关性。 图4a显示了一个代表性的结果不正确执行一个实验。两个对照组（黑网吧）和转染细胞的分子伴侣（红色条）没有显示增加蛋白质复性时间。此外，转染的分子伴侣复性的增加并没有导致。 图证实这种相关性较差。 4B显示控制（黑线）和转染细胞的分子伴侣（红线）的R平方值分别为0.6619和0.1882。

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作者什么都没有透露。

在这项工作中来衡量细胞内分子伴侣复性活动的协议。整个实验可在3到4天，在图概述所示。 1。 萤火虫和海肾荧光素酶产生的光信号的鲁棒性和线性代表一个坚实的基础协议的重现性。 检测的关键步骤是一个有效率的转染试剂的选择，以确保分子伴侣和萤火虫荧光素酶的过度表达。报告基因可交付也与其他方法，如腺病毒?...

在这个实验中使用一个光度计形式Perkin Elmer公司&#39;1420发光反“矢量之光” 方案的光度计： 海肾：100μL注射泵1 反应缓冲液：Pump1，70μL注入荧光素：Pump2，30μl注入对于缓冲区准备在双蒸水，这些股票的解决方案： <ul style="list-style:none"><li> - 1M 硫酸镁 ：246,48克硫酸镁4 * 7H 2 O在1公升</li><li> - 0.5米EGTA：190,18克EGTA 1升</li><li> - 1M KH 2 PO 4：136,09克KH 2 PO 4 1升</li><li> - 1M K2 HPO 4：228,23克K 2 HPO 4 3H 2 O在1公升</li><li> - 5M的 NaCl：292,2 g氯化钠1升</li><li> - 0.5米EDTA：186,12克EDTA · 2H 2 O在1公升&nbsp;</li></ul>甘氨酸 - 甘氨酸缓冲： <ul style="list-style:none"><li> - 25MM Glycylglycin </li><li> - 4硫酸镁15MM </li><li> - 4MM EGTA </li></ul>对于1公升使用3,3克Glycylglycin，一个1M的硫酸镁溶液和一个0.5米EGTA解决方案8毫升15毫升。解决水，调节pH值7,8和带来一个总体积为1升。 海肾/腔肠缓冲区的反应缓冲液： <ul style="list-style:none"><li> - ＆NBSP; 13.4毫米KH 2 PO 4 </li><li> - 86,6毫米KH 2 PO 4 </li><li> - 0,5 M氯化钠</li><li> - 1mm的EDTA </li></ul>对于1升使用13.4毫升一个1M KH 2 PO 4溶液中，一个1M K 2 HPO 4溶液，100毫升一个5M的NaCl溶液和一个0.5米的EDTA溶液2毫升86,6毫升。 <table border="1"><tr><td></td><td> 公司 </td><td> 目录/产品编号： </td></tr><tr><td> DMEM（贝科的修改鹰中等1X） </td><td> GIBCO公司</td><td> 41966029 </td></tr><tr><td> 胎儿牛血清黄金 </td><td>临时机场管理局</td><td> A15 - 151 </td></tr><tr><td> 9基因的X极端 DNA转染试剂</td><td>罗氏公司</td><td&gt; 06365809001 </td></tr><tr><td> PBS 贝科的磷酸盐缓冲盐水1X </td><td> GIBCO公司</td><td> 14190-094 </td></tr><tr><td> MOPS 3 - （N -吗啉）丙磺酸</td><td> Sigma - Aldrich公司</td><td> M1254 </td></tr><tr><td> 放线菌酮 </td><td> Sigma - Aldrich公司</td><td> C7698 </td></tr><tr><td> 被动裂解缓冲5X </td><td> Promega公司</td><td> E194A </td></tr><tr><td> Glycylglycin EGTA 硫酸镁 （硫酸镁4 * 7H 2 O） </td><td> Sigma - Aldrich公司 罗斯 罗斯</td><td> G7278 3054.2 P027.1 </td></tr><tr><td> KH 2 PO 4 K 2 HPO 4（K 2 HPO 4 * 3H 2 O） 氯化钠 EDTA </td><td>罗斯 罗斯 罗斯 罗斯</td><td> 3904.1 6878.2 3957.1 8043.1 </td></tr><tr><td> 荧光素萤火虫 </td><td> Biosynth </td><td> L8200 </td></tr><tr><td> 腔肠 </td><td> Biosynth </td><td> C7000 </td></tr><tr><td> DTT（二硫苏糖醇） </td><td>罗斯</td><td> 6908.2 </td></tr><tr><td> ATP（三磷酸腺苷） </td><td>罗氏公司</td><td> 10519987001 </td></tr></table>

intracellular refolding assay

本协议描述了一个方法来衡量，在基于单元的系统和抑制/刺激活性的化合物可能产生的影响酶的活性分子伴侣。分子伴侣是参与调节蛋白质折叠1的蛋白质，有促进细胞存活时强调侮辱，如热休克2，营养饥饿和接触化学品 /毒药3的关键作用。出于这个原因的伴侣被发现在参与活动，如肿瘤的发展，癌细胞4 chemioresistance以及神经退行性疾病5。能够抑制或刺激这些酶的活性小分子的设计，因此，用于癌症治疗和神经退行性疾病的研究最多的战略之一。这里所描述的实验提供的可能性来衡量一个特定的分子伴侣复性的活动，并研究对比赛的影响ounds就其活动。这种方法调查的分子伴侣基因转染的萤火虫荧光素酶基因的表达载体。它已经描述，可以通过10,11分子伴侣复性变性萤火虫荧光素酶。作为转染控制正常化，为海肾荧光素酶基因的载体转染。在本协议中所述的所有转染基因的X极端 11（罗氏）在HEK - 293细胞。在第一步中，蛋白质的合成，抑制细胞治疗与放线菌酮。此后蛋白展开是在45 ° C诱导热休克30分钟。恢复后，在37 ° C，蛋白质重新折叠成其活性构象和萤火虫荧光素酶的活性是用作读出：会产生更多的光，更多的蛋白质将重新获得原来的构象。非热震惊细胞作为参考（100％的复性荧光素酶）。

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Intracellular Refolding Assay

在这个协议的方法来衡量热休克后细胞内蛋白质复性的描述。这种方法可以用来研究如分子伴侣和他们的共同因素或化合物能够影响他们的活动foldases。萤火虫荧光素酶的活动是作为记者来衡量伴侣复性活动。

细胞内复性检测

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

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Biology

14.1K 次观看.  Karlsruhe Institute of Technology. 在这个协议的方法来衡量热休克后细胞内蛋白质复性的描述。这种方法可以用来研究如分子伴侣和他们的共同因素或化合物能够影响他们的活动foldases。萤火虫荧光素酶的活动是作为记者来衡量伴侣复性活动。

视频: Intracellular Refolding Assay

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

成像APP的细胞内运输与光活化GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

纯化及复性的淀粉样纤维（他）<sub&gt; 6</sub&gt; -tagged重组Shadoo蛋白表达的包涵体<em&gt;大肠杆菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...