这项工作是由全国健康教育资助EY0081和5T3​​2UY07145研究所和圣殿骑士眼科基金会的支持。贾森Rihel是一个生命科学研究基金会的布里斯托尔 - 施贵宝研究员。

该协议列出的步骤来执行在自己的实验室光增量和递减的斑马鱼幼虫的视觉运动反应。斑马鱼是一个伟大的行为研究模型系统。他们易于维护，他们有很大的离合器大小，并迅速发展。例如，斑马鱼幼虫的眼睛是顺应轻发展的第3天，在这时候，他们表现出了惊吓反应。 第1部分：电镀成96孔板个别鱼<ol><li> WT幼虫生长在一个黑暗/光周期在28 ° C，直到受精后至少4天（DPF）。我们典型的光：暗周期是14小时的灯光，在上午9:00开始，10小时的灯关闭，下午11:00开始。为了获得最佳的行​​为结果，避免过度拥挤;我们通常不超过50幼虫保持在一个培养皿。 </li><li> 4 DPF后，斑马鱼是准备转移到一个96孔板。为了让更多的幼虫游泳室，我们通常使用一个96孔板，650μL以及大小与大，但是，标准的96孔板的工作只是罚款。使用的塑料转让吸管，轻轻地转移每幼虫。 </li><li>转移入井的鱼后，填写水面近冲洗井的顶部有足够的鱼水之每口井。溢出或底部填充的以及可能会导致录制相机的光学问题。此外，小心不要引入气泡入井。 </li></ol>第2部分：调查的录音装置 对于这个协议的一部分，请参考电影识别组件，并熟悉我们设置录音装置自己。 <ol><li>录音室里面是一个定义良好的位置的96孔板。 </li><li>相机是定位在后面，重点是对使用过的框镜板。这些反射镜的角度可以调整，通过旋转螺丝固定到位的镜子。 </li><li>录音室是从底部通过红外发光二极管照亮。这使得相机即使在黑暗中看到的鱼。幼虫无法检测到红外光，所以这个常数的红外照明，不影响实验。白光LED，也照亮从下面的录音室。他们分别从红外灯控制。照明的白光从上面或从侧面室当然是可能的，然而，在这些情况下，必须采取离水面，可能会干扰相机，避免强烈刺眼。 </li><li>对于实验，超过几个小时，填室轻轻运行的水，以帮助保持恒温实验的持续时间。完成的水不断流的一种方法是由一个小型的水族馆泵，是一个典型的水下水族馆加热器加热至28 °的水从水库泵。 </li><li>为了尽量减少杂散振动从房间，整个录音单位应当搭起一个沉重的余额表。 </li></ol>第3部分：与电脑的视频跟踪电网的96孔板中的对齐方式 软件 <ol><li>将96孔板中含有鱼进录音室。 </li><li>使用水浴时，慢慢地在水中放置板，水位有机会的情况下调整蔓延到盘子。另外，关闭水流，加板，然后恢复流动。此外，请务必使用一个弹簧或橡皮筋​​固定到位96孔板。 </li><li>的观点Videotrack软件，检查您的实验，所有的幼虫在电脑屏幕上是可见的。使用该软件的控制，配合你的盘子里，这样，每条鱼是在一个正方形网格井，视频跟踪软件的电网。跟踪计算机将计算运动分别为这些框，所以如果你错位的计算机网格，一些鱼的动作可能会丢失。或者更糟糕的是：两个相邻的鱼会占用同一地区，并作为一条鱼算。为您的录音，这一步是非常重要的的。 </li><li>对齐后，程序应该去当灯光开启和关闭的时间。我们通常允许3个小时的浅色或深色的包装盒中的适应，既要获得一个基线活动水平，但也给幼虫有机会冷静下来以下的移液器和处理。基线后，我们再备用开​​灯关灯30分钟与30分钟，并重复数次。 </li><li>下一步，关闭录音室的门，并开始录制。 </li><li>在实践中，我们记录每个每秒鱼的活动，但实际上视点软件记录帧数据的帧（指电影数据收集示范）。设置每帧的最小像素变化的阈值将一定程度上取决于您的特定的摄像头和光线设置。我们的设置，我们通常使用的4个像素的门槛，也就是说，如果少于4个像素发生变化，它被认为是背景。如果超过4个像素的移动，则表示鱼是移动。我们凭经验确定，截止检测几乎所有的幼虫游泳和转身动作。 </li><li>即使录音盒隔离幼虫相当不错，关闭在房间里的光线和照顾，以尽量减少机械噪声，例如关闭和打开房间的门，有一个舞会，或做你的锻炼例程中断。 </li></ol>第4部分：数据分析<ol><li>实验完成后，转移到一个Excel工作表或到您最喜爱的分析套件收集的数据。 </li><li>所附的Excel工作表是一个什么样的数据可能看起来像的例子：它包含在几秒钟内从每人幼虫秒，所有的鱼在整个实验的实验和活动的开始时间（见补充文件的示例数据文件的节本页面）。 </li><li>图1显示了一个单一的幼虫活动的一个例子。图2是一个代表40 WT斑马鱼幼虫的平均跟踪。平均和关闭的反应是突出和一致的。 <img src="/files/ftp_upload/old/923_2008_9_26_9/923_Figure-02.png" alt="图1" width="569" height="398" /> 图1：5 DPF一个单一的聚鱼一条鱼的活动在光周期的30分钟交替的ON和OFF 活动 。对反应表示黑色箭头和红色箭头OFF反应。 <img src="/files/ftp_upload/old/923_2008_9_26_11/923_Figure-03.png" alt="图2" width="577" height="387" /> 图2：40鱼的平均活动40 5 DPF WT鱼在光周期的30分钟交替的ON和OFF平均活动 。平均（黑色箭头）和OFF（红色箭头）的反应是突出和一致的。 </li></ol> 5。代表性的成果图3是一个我们所有的实验中所用的实验大纲的原理轮廓。 <img src="/files/ftp_upload/old/923_2008_9_26_12/923_Figure-04.png" alt="图3" width="530" height="479" /> 图3。实验设计的视觉运动反应器（VMR）测试。）鱼被放置在一个96孔板，在录音室。每条鱼的活动，是衡量每秒。 B）鱼都获得了或深或浅的适应期，解决他们获得基线活动水平。期间关闭30分钟的灯和灯光是连续推出了3个小时的总。此图是改编自探针等，2006年。 视觉运动反应图什么样子呢？ 我们测得的聚鱼灯的递增和递减的ON和OFF反应。为了证实这些反应依赖于眼功能，我们测量CHK突变体的活动，不发展任何眼睛。图4显示了从野生型动物以及CHK突变体获得的平均活动。 <img src="/files/ftp_upload/old/923_2008_9_26_13/923_Figure-05.png" alt="图4" width="489" height="212" /> 图4：WT鱼有明确的ON和OFF斑马鱼幼虫在5 DPF光对30分钟和30分钟的灯熄灭的运动行为是由眼睛的外侧介导的反应是每秒记录。每个跟踪代表平均480从120个人WT（蓝色线）或CHK突变的幼虫（橙色线）录得超过3实验的反应。 CHK突变体没有显着增加他们的活动，无论是轻的递增或递减，有一个活动的基线水平低。改编自Emran等人，2007年这个数字。 从NRC突变鱼的视觉运动反应。 NRC的突变被认为是完全失明OKR测试的基础上。 NRC的突变体，感光终端不正确形式和视觉通路上却是大打折扣 1 。从这些突变鱼视网膜神经节细胞的录音显示，他们表现出断型神经节细胞的反应，一些异常的ON - OFF，但没有纯粹的 2型反应为主。 我们使用VMR测试表明，NRC突变正常关闭响应延迟和缓慢的响应（ SEE图5）。因此，NRC的突变并非完全失明以前认为的2。 <img src="/files/ftp_upload/old/923_2008_9_26_14/923_Figure-06.png" alt="图5" width="591" height="244" /> 图5：NRC mutats在光照强度的变化增加其活动的行为反应，指示灯亮起，WT 和 NRC突变幼虫在5 DPF 。每个跟踪代表平均480 120名来自各基因型的个体鱼的反应。 NRC突变体的平均运动器官的行为（粉红色的痕迹）相比，聚鱼（蓝色的痕迹）略有减少，但仍然旺盛关刺激。注意在NRC突变响应缓慢的上升时间亮起比较轻的WT鱼响应。

<ol>
	<li>Allwardt, A. B., Lall, B. A., Brockerhoff, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+formation+is+arrested+in+retinal+photoreceptors+of+the+zebrafish+nrc+mutant.">Synapse formation is arrested in retinal photoreceptors of the zebrafish nrc mutant.</a> J Neurosci. 21, 2330-2330 (2001).</li><li>Emran, F., Rihel, J., Adolph, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OFF+ganglion+cells+cannot+drive+the+optokinetic+reflex+in+zebrafish.">OFF ganglion cells cannot drive the optokinetic reflex in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 19126-19126 (2007).</li><li>Prober, D. A., Rihel, J., Onah, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocretin/orexin+overexpression+induces+an+insomnia-like+phenotype+in+zebrafish.">Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish.</a> J Neurosci. 26, 13400-13400 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish. , (2000).</li></ol>

我们在影片中展示的实验程序是代表所有的WT鱼。然而，这些实验可以做类似于突变鱼（见代表结果部分）。我们建议板聚鱼和在棋盘大纲为最优控制的目的相同板上的突变鱼。 当使用相同的板聚鱼突变的鱼，确保您写下什么类型的鱼，每孔镀。这里有一个如何保持鱼镀在每口井的轨道（显示数据表的注意书）的建议。 另外，最好在一天的同一时间做了一定的实验重复。例如，如果你开始的3个小时的...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Microplate devices</td>
<td>Tool</td>
<td>Whatman</td>
<td>7701-1651</td>
<td>&nbsp;</td>
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<td>Transfer pipetes</td>
<td>Tool</td>
<td>VWR</td>
<td>202205</td>
<td>&nbsp;</td>
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<td>Fish water</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>refer to reference #4</td>
</tr>
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<td>Recording chambers (Zebrabox)</td>
<td>Tool</td>
<td>Viewpoint Lifesciences</td>
<td>&nbsp;</td>
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<td>Videotrack Software</td>
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a behavioral assay to measure responsiveness of zebrafish to changes in light intensities 

视动反射（OKR）是一个基本的视觉反射展出，大部份脊椎动物和眼视觉场景相对稳定起着重要作用。然而，OKR需要检测的动物移动的条纹，它可能是鱼类，未能表现出一种OKR未必完全失明。斑马鱼突变体，没有视动反应彗星（NRC）在任何光线条件下测试，并报告完全失明没有OKR。此前，我们已经表明，OFF神经节细胞的活性，可在这些突变体的记录。为了确定是否与没有NRC的突变体，如OKR突变鱼可以检测到简单的轻的递增和递减，我们开发的视觉电机行为检测器（VMR）。在这个实验中，单斑马鱼幼虫放置在每个允许同时使用自动视频跟踪系统的幼虫监测的96孔板以及。每个幼虫的30分钟指示灯亮起，指示灯熄灭30分钟的运动反应记录和量化。聚鱼后灯被称为惊跳反应，随后返回到低比基线活动电机活动的一个简短的扣球，被称为冻结。聚鱼也大幅增加他们的自发活动紧随灯光熄灭，只有逐步在几分钟内恢复到基线自发活动。 NRC的突变体的回应同样光聚鱼关，但表现出他们的平均活动为WT鱼相比略有减少。电机响应活动光NRC突变延迟和缓慢。有一个NRC的突变响应缓慢的上升时间，光相比WT光响应。结果表明，NRC鱼是不完全失明。因为硬骨鱼类可以检测到通过非视网膜组织中，我们确认使用chokh（CHK）的突变体，完全没有眼睛，从发展的最初阶段，直接的行为反应的光强度的变化需要的完整的眼睛。在我们的VMR检测，CHK突变体表现出没有惊吓反应，无论是轻或关，表明横向眼睛调解这种行为。 VMR检测形容这里补充行之有效的OKR检测，不测试斑马鱼幼虫的能力以应对光强的变化。此外，VMR检测的自动化，适合于高通量筛选驱动的视觉反应的光强度的缺陷。

Watch this Scientific Journal Video about A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities at JoVE.com

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

我们开发的视觉运动反应，反应轻的递增和递减定量幼虫斑马鱼的电机输出。我们还研究了斑马鱼的视觉突变，包括没有视动反应（NRC）的突变体，它被认为是完全失明的另一个视力检测，视动反射测试时。

斑马鱼的响应行为的检测，以测量光强的变化

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

a-behavioral-assay-to-measure-responsiveness-zebrafish-to-changes

Research

JoVE Journal

Biology

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

16.2K 次观看.  Harvard. 我们开发的视觉运动反应，反应轻的递增和递减定量幼虫斑马鱼的电机输出。我们还研究了斑马鱼的视觉突变，包括没有视动反应（NRC）的突变体，它被认为是完全失明的另一个视力检测，视动反射测试时。

视频: A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

科研

生物学

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

使用拉姆齐试验来测量液体分泌和离子流率在<em&gt;果蝇</em&gt;马氏管

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...