经费是由美国威斯康星州发现的研究所。

在这份报告中，我们展示了一个流体输送的方法，利用小液滴表面张力泵通过一个微流体通道所需的量，以达到不同的流体现象。例如，用户可能希望尽可能快流单液，或在连续快速创建特定的流体模式提供多种流体。为了做到这一点，用户必须首先有一个微流体装置周围建立了一个应用程序。 microflluidic设备并不需要保税，但应该从一个亲水性材料。 Therfore，该方法可以利用几乎所有的微流体装置，性能在很大程度上微流体通道的几何约束的要求。为了帮助浏览此方法，介绍了相关的数值分析的几何约束。 <ol><li> 分析方法：根据拉普拉斯法和沃什伯恩法[1]，可以与在微流体通道，其尺寸和流动的液体，如公式（1）属性的流量， <img src="/files/ftp_upload/1329/eqn1.png" alt="式（1）" /> （1） ΔP是入口和出口之间的压力差，γ是液体的表面张力，R是进口下降半径，Q是流量和 K是由方程（2）中所描述的流体阻力， <img src="/files/ftp_upload/1329/eqn2.png" alt="公式（2）" /> （2） 其中，η是液体的粘度，L 0是渠道的长度，H为通道高度，W为信道宽度，λ= W / H 和G（λ）= 1.5，λ&gt; 4.45或 <img src="/files/ftp_upload/1329/g_lambd.png" alt="符号6" /> 如果λ&lt;4.45。方程（2）代入式（1），总是假定H &lt;W和解决的Q，可以得到式（3）， （3） <img src="/files/ftp_upload/1329/eqn3.png" alt="等式（3）" /> 同样的分析可以知道一个通​​道内流体的速度为Q = VA，其中V是流体的平均流速和A是截面积或硬件。插入这些方程（3）你想出方程（4）， （4） <img src="/files/ftp_upload/1329/eqn4.png" alt="方程4" /> 经常应用在微流体生物的一个重要的机械概念，即是剪应力，这涉及到流率和速度，由式（5）， （5） <img src="/files/ftp_upload/1329/eqn5.png" alt="公式（5）" /> 明知流量，流速和渠道尺寸和流体性质的功能，其物理意义之间的关系是在一个微流体装置的设计对于一个给定的目的是至关重要的。一旦创建一个设备，用户必须校准流体输送系统设备内达到所需的流量特性。 </li><li> 在设置和校准配送系统的步骤： <ol><li>通过软光刻技术，采用聚二甲基硅氧烷（PDMS，Sylgard 184，道康宁）[2]微流体装置。有朱庇特的文章，说明制作的PDMS微流体装置[5]的方法。这个演示中，我们选择了一个简单的直连通道，，尺寸如下：2.2毫米宽，10毫米的长度和260um高度。入口和出口直径1.8毫米和5.1毫米（图1）。可逆PDMS的设备连接到玻片上按到玻片上（或其他合适的基板），挤掉任何气泡[5]。一个可逆的附件允许设备重复使用多次。该方法也可用于与永久保税设备，但它不是必需的。 </li><li>装满液体的设备。的硅橡胶的疏水性和亲水性玻璃帮助一个放置在进口下降，出口或移动，进入通道。如果液滴不希望通过自身的渠道，或如果气泡进入通道移动进入，用户可以在入口或出口的液滴，并在另一端使用一个吸管吸吮的液体通过渠道。帮助进入通道的液动的另一种方法是从载玻片上分离的PDMS设备，并轻轻清洗用乙醇的PDMS设备和载玻片。这PDMS和载玻片上的疏水和亲水的性质，分别，这可能会随着时间的推移和使用已减弱回报。 </li><li>与液体灌装设备后，地方上的进气口的小水滴和比格ř在出口下降。确保被动抽水正在发生的事情通过观看入口崩溃的小水滴，观察液体流量对出口。再次，确保通道内无气泡。 </li><li>使用的利公司的[3] VHS微配药开始套件，放在一起的一个或多个阀门（阀图2设置）的李VHS M / 2 24伏阀组成，一个0.062与孔口的0.0100大小MINSTAC喷嘴“，在李0.062 Minstac软管适配器，李穗和保持驱动器（用户控制，未显示）和引线大会（阀门的连接穗，并保持驱动器，不显示）。 </li><li>按住阀门的一种简单方法是使用生物科学工具微型人（图2）[4]。这提供了一种方法，精确瞄准和保存在某个位置的阀门，在阀门粘合实验持有人的一端，并在另一边使用一个磁性底座（未显示）。 </li><li>请几英尺以上的PDMS微流体装置（在我们的例子中，我们使用¾每盎司注射器开放的环境，见图2）的水库系统。该水库提供了一个压头驱动喷嘴，与水库的高度成正比的压力。另外喷嘴阀可加压任何不同的方式（即压缩天然气）。将注射器针头注射器。一个典型的注射器针头将很容易附着到1.14毫米内径管。在1.14毫米的油管，然后轻松地附加到1.58毫米（1 / 16“）内径管，然后本身连接到”软阀门管的适配器“。为了防止泄漏液体在1.14毫米至1.58毫米的管道连接，可以使用硅橡胶密封剂。现在有一个注射器针头和利有限公司阀之间的线，填充液体注射器水库。额外的注射器和一个阀门，可以用来帮助在清洗过程中（在图2所示，但不标记）。阀门一侧放置一个磁铁，这是如何将这些阀门被清除（他们常闭电磁阀），并观看流经阀门和出0.0100 &#39;&#39;喷嘴的液体开始从水库。 </li><li>选择一个阀门开放时间（开放时间是阀允许液体通过对每一个脉冲的基础上的时间）和频率（每秒的脉冲数）的校准系统。激活一个选定的时间（一分钟左右，只记得总运行时间）阀。称取液体从阀门交付。了解总的运行时间，频率和每个脉冲的开放时间，计算每毫秒克拍出来阀门。这种“克每毫秒”的价值将允许你选择任何想要的音量，用户可能希望从阀门交付的开放时间。 </li></ol></li></ol>例：一分钟（60秒）激活系统。频率为15赫兹（15脉冲在一秒钟内）。每个脉冲的开放时间为20毫秒（ms）。 （20毫秒）（15Hz的）（60秒）= 18000ms。 这意味着，在一分钟内60000 MS中，阀门实际打开为18000毫秒。 假设流体的交货量重达5克。然后， 5克/ 18000毫秒= 2.78e -4克/ MS。 在水的情况下，其密度为克每毫升（ML）， 2.78e -4克/ MS = 2.78e -4毫升/ MS。 校准后，下降的体积是依赖于开放时间。例如，开放时间为20ms，和所有参数保持在前面的例子一样， （2.78e -4毫升/ MS）（20毫秒）= 5.56e -3毫升= 5.56μL。 要查找的开放时间y需要作出的X微升（微升）量下降， （x微升）/ [（2.78e -4毫升/ MS）（1000μL/毫升）] = Y毫秒 8）瞄准一个或多个喷嘴的PDMS设备的进气口（图3）。经校准系统，走出来，每个阀门，基于微流体装置尺寸计算体积。对于高速被动抽水（获取最大流速），计算进滴体积要创建一个入口下降入口面[2]拥有一个90度接触角。对于分组创作，计算阀频率和开放的时代，要激活序列在两个阀门的阀计时的。正如图3可以看出，两个喷嘴可指出在入口处。这可以扩展到多个喷嘴，所有目的都在于在通道入口处。 代表性的成果： 正确校准时，阀门打开，正确计算时间和正确的入口的喷嘴，用户应该能够看到被动泵的流量（图4）。一阵液体SH乌尔德出来的阀门，到达入口。液体到达入口，进入通道的入口下降有一个瞬间崩溃，对插座。通道内的液体移动只能在一个进口下降崩溃。完整的通道内流体运动停止在下拉倒塌年底，瞬时流体停止提供和定义良好的流体边界的情况下，用户是流动的多种液体。下降崩溃的持续时间取决于进气口半径和体积的进口下降[1]。在我们的实验装置和设计中，进降塌陷发生在几毫秒的问题。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1329/figure1.jpg"><img border="0" src="/files/ftp_upload/1329/figure1tmb.jpg" alt="图1" /></a> 图1。PDMS微流体装置与一个入口，左，和一个电源插座，右。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1329/figure2.jpg"><img border="0" src="/files/ftp_upload/1329/figure2tmb.jpg" alt="图2" /></a> 图2水库系统和阀门安装。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1329/figure3.jpg"><img border="0" src="/files/ftp_upload/1329/figure3tmb.jpg" alt="图3" /></a> 图3。两个阀门，旨在在一个微流体装置的单进。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1329/figure4.jpg"><img border="0" src="/files/ftp_upload/1329/figure4tmb.jpg" alt="图4" /></a> 图4。时间步长的序列（33毫秒），进口下降崩溃的液体从阀门的弹射。

<ol>
	<li>Berthier, E., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+rate+analysis+of+a+tension+driven+passive+micropump.">Flow rate analysis of a tension driven passive micropump.</a> Lab Chip. 7, 1475-1478 (2007).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Prototyping+of+Microfluidic+Systems+in+Poly(dimethylsiloxane).">Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).</a> Anal. Chem. 70, 4974-4984 (1998).</li><li>Harris, J., Lee, H., Vahidi, B., Tu, C., Cribbs, D., Cotman, C., NL, J. e. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-plasma+Bonding+of+PDMS+for+Inexpensive+Fabrication+of+Microfluidic+Devices.">Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices.</a> J Vis Exp. (9), (2007).</li><li>Walker, G. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+pumping+method+for+microfluidic+devices.">A passive pumping method for microfluidic devices.</a> Lab Chip. 2 (3), 131-134 (2002).</li></ol>

<ol><li>对于高速抽水，如果选择正确的组合（由于正确的开放时间）的频率和脉冲量每被动，用户应该看到什么似乎是一个静态的下降或壳入口和通道内的流动速度非常快。如果发生溢出，开放时间和/或频率过高。 </li><li>要检测的势头/表面张力的相互作用，用户应在泵一个脉冲，而脉冲发生（从开始到结束），观察通道内的环境。作者建议使用荧光珠，提供了一个通道内流体行为的准确描述。一个单一入口下降?...

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<td>Sylgard 184 Silicone elastometer base</td>
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high speed droplet-based delivery system for passive pumping in microfluidic devices

一种新型的微流体系统已经开发使用的被动抽水用户控制液滴基于流体输送系统沿现象。被动式抽水，是由表面张力引起的压力差驱动流体运动的现象在封闭的渠道。自动化流体输送系统由电压控制阀，微流体水库和控制系统连接到喷嘴。这些电压的控制阀，提供了一个容积精确的方式提供高频率地在微流体装置的入口流体液滴。在目前的研究例子展示了尺寸的基础上，该系统能够流动每分钟4毫升（通过260um截面渠道2.2毫米）。根据这些相同的通道尺寸，通道内的流体交换可以实现低至8毫秒。据观察，有动力系统（在通道的阀门和流体的速度创造了水滴的组合传授），液体的表面张力之间的相互作用。势头提供了速度，流体的流量（或反之亦然），在入口处的表面张力的平衡提供了任何流量的突然停止。这突然停止，允许用户控制通道的流量特性和各种生物应用打开门，不等药物细胞研究试剂交付。它也观察到，喷嘴的目的是在浅层的角度入口时，液滴的势头可能会造成额外的有趣流体现象，如在多个入口液滴混合，。

Watch this Scientific Journal Video about High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices at JoVE.com

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

一种新型的微流体系统已经开发使用的被动式抽水的现象和用户控制流体输送系统。这种微流体系统已在多种生物应用由于其成本低，易于使用，体积精度高，速度快，重复性和自动化的潜力。

基于液滴在微流体装置的被动抽水高速输送系统

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

high-speed-droplet-based-delivery-system-for-passive-pumping

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.7K 次观看.  University of Wisconsin-Madison. 一种新型的微流体系统已经开发使用的被动式抽水的现象和用户控制流体输送系统。这种微流体系统已在多种生物应用由于其成本低，易于使用，体积精度高，速度快，重复性和自动化的潜力。

视频: High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

PDMS微流体装置的廉价制造的非等离子键合

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

微流控一次性诊断的应用

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...