这项工作是由来自美国国立卫生研究院（ES07408和EB00208）的赠款支持。

1。 ECIS的/出租车电极的制备<ol><li> ECIS的/的士电极的阵列（8％幻灯片商会组成）的金表面稳定在室温为15分钟，在无菌条件下用无菌去离子水（DH 2 O）的10毫米半胱氨酸的前处理。 </li><li>从每个电极室吸半胱氨酸溶液，冲洗与消毒卫生署2 O的 3倍，并更换完全培养基250μL（RPMI 1640培养液，10％胎牛血清，25毫米的HEPES缓冲液）。 </li><li>连接电极阵列联系仪器阵列支架引脚进行电极检查。 </li><li>正确连接钱伯斯将在绿色突出显示在电脑屏幕上。如果商会以红色突出显示，它们没有连接电极阵列持有人应重新定位，以建立各商会的接触。 </li><li>所有商会一旦连接后，点击"检查"按钮，在软件控制器，以防止在雷初阻力为每个室和电容值。电阻值应该是在1600年和2000年之间欧姆，电容值应该是一个8腔阵列5和6之间的纳法。如果没有在这些接受的参数，一腔室个别不应该被用来收集数据。 </li><li>电极断开阵列持有的准备个别的琼脂糖商会。吸商会介质和清洗每个室用无菌DH 2 O 3倍</li></ol> 2。准备琼脂糖钱伯斯<ol><li>在15毫升无菌聚丙烯锥形管，措施0.03 GTG琼脂糖Seakem g和添加1毫升无菌PBS。更换管帽松散，融化在高压灭菌器为2分液循环琼脂糖（微波炉融化此法有效）。这短暂的时间，不会融化的锥形管。对于不太讲究的细胞，不需要血清文化，（如粘菌 ）0.5％琼脂糖凝胶可在适当的介质，在微波炉中融化。反应釜需要血清的细胞是必要的，因为这将变性如果在微波融化。 </li><li>完整的RPMI水浴中预热的培养基中加入5毫升（55°〜60°C）的直接热琼脂糖的终浓度为0.5％琼脂糖和吸管了下来，轻轻地混合。 </li><li>吸取300μl琼脂糖融化到每个室的中型的，取代ECIS的/的士阵列盖，并允许在室温下的凝胶。 </li><li>吸取剩余琼脂糖到15毫升管帽评估琼脂糖凝胶。这样，多余的琼脂糖也将被用来在第4部分:切割井到琼脂糖。 </li></ol> 3。建设和锐化套管井刀具<ol><li>使用5/64"位。旋转的钻头，在每一个低速钻套管开幕尖端，使钻头降低整个室内每个套管运边缘锐化两个14针的钝端套管，ening形成一个锋利的切削刃（ 图4A）。 </li><li>通过1"×2"×¼"有机玻璃（ 图4B - D）与5/64"位在钻床钻两个孔。插入每个削尖的14计套管，通过每个有机玻璃洞的距离相等，所以它突出。 </li></ol> 4。到琼脂糖切割井<ol><li>轻轻燃烧的秘诀，消毒14号套管。 ECIS的商会在切割井前，允许套管冷却至室温，或冷却触摸琼脂糖在15毫升管帽的套管。如果套管的温度过高，会熔化琼脂糖室井被切断，使井的畸形和缺陷。 </li><li>将套管对上述两个黄金点周围的电极（ 图2c和5）在每个室的目标。插入导管垂直之间的套管和温柔的接触后，没有任何横向移动，停止在电极表面。小心取出套管，再没有任何横向运动。额外井反对梯度安置，也可以用套管夹具的重新配置。 </li><li> 轻轻吸套管留下琼脂糖插件，使用无菌9"巴斯德吸管，连接到低真空压力，真空陷阱捕捉琼脂糖浪费。 </li><li>加入300μL完整的RPMI媒体表面各室在37°C间，2分钟到饱和的琼脂糖，然后轻轻地吸不干扰琼脂糖从表面和媒体的水井。 </li></ol> 5。载入井<ol><li>每口井是持有7微升量的能力。在每个室以及一，增加7μL的细胞悬液。对于Jurkat T细胞，使用2×10 7细胞/ ml。这些细胞会回应7μL的200毫微克/毫升SDF-1α在不完整的RPMI稀释，缺乏血清。使用完整的媒体单独作为一个负续列伊。 </li><li>加载井后，倒置显微镜查看每个阵列室，以确保所有井被切断，并填写正确。细胞应该是很好的边界​​的范围内。如果他们看到在两个井，或超出细胞以及边境蔓延，已形成一定的差距下的琼脂糖。注意设置不当的井，数据不能可靠地解释这些商会。 </li></ol> 6。数据采集<ol><li>将电极阵列中的持有人，并重新连接电极阵列上的金片，弹簧加载的POGO针持有人，并单击"设置"。井，已正确连接，将突出绿色。如果该井是红色的，它们没有连接和电极阵列持有人应重新定位。 </li><li>软件面板中，从下拉菜单中选择"8WE1"（8以及1电极）来表示阵列配置。 </li><li>选择"检查"软件CONTROL面板确定为每口井的初始电阻和电容值。电阻值应该是在1600年和2000年之间欧姆，电容值，而应该是5和6之间的纳法。 </li><li>选择多个频率在几个频率来衡量的阻力。定制频率，或使用了一套推荐的频率有一个选择。标准的多频率采集设置捕获数据，在11个预先设定的频率在8孔/分钟的速度（52.5，125，250，500，1000，2000，4000，8000，16000，32000和64000赫兹）。也可以自定义的测量之间的时间间隔。多频数据收集允许电容，阻抗和阻力分析。收集在4000赫兹的电阻值通常是足够的哺乳动物细胞趋化研究，但可提供额外的频率在12,13其他情况下的信息。 </li><li>总的实验时间可以设置，右手手动面板，可以停止实验，一旦细胞产生的阻力，表明在到达电极，通过点击"完成"。细胞到达电极所花费的时间取决于所用的细胞类型。 </li><li>点击"开始"启动数据收集。 </li></ol> 7。数据管理<ol><li>虽然可以将数据导出到Excel，ECIS的1600R软件，其中伴随ECISθ系统是最有效的方法分析ECIS的/出租车数据。 </li><li>软件的数据分析工具栏包含的单选按钮，Z，R和C，这些决定是否阻抗，电阻，电容，分别对x轴（经过时间）垂直轴绘制。 </li><li> ECIS的/出租车数据显示，随着时间的推移阻力位在4000赫兹是最有效的。快速波动性，或microtransients（ 图1），在电极细胞运动的证据。编译，Oŕ分级的数据点，因此，并非所有的点绘制，不建议ECIS的/出租车，因为它的数据平均microtransients。 </li><li>一旦图已与ECIS1600R软件，它可以从文件菜单中选择"出口图"，并保存。tif或jpg文件导出。 </li></ol> 8。代表结果细胞趋增加，阻力位在4000赫兹表示。到达目标电极上的细胞也被称为microtransients，或微动，奥普等阻力迅速波动的外观表示。人，2009年（ 图1）14。阴性结果表示一致，或略有下降，在4000赫兹电阻，在红色如图1所示 。没有microtransients也是一个电极细胞运动的情况下的一个标志。在阻力增加是直接亲到细胞交叉电极10的数量成正比。单克隆抗体除了特定的趋化因子或毒素已知的干扰与G蛋白偶联受体可以阻断趋化11。 <img alt="图1" src="/files/ftp_upload/3840/3840fig1.jpg" /> 图1。人Jurkat T细胞趋化反应SDF1α人 Jurkat T细胞（2.0x10 6细胞/毫升）被暴露在SDF-1α梯度（起始浓度为100毫微克/毫升）或RPMI 1640培养液作为阴性对照。在4000赫兹的规范化阻力绘制。人类Jurkat T细胞移动响应SDF-1α，增加阻力和microtransients证明，没有动静，而被曝光的RPMI 1640。 <img alt="图2" src="/files/ftp_upload/3840/3840fig2.jpg" /> 图2。顶视图的ECIS的/出租车电极withouţ琼脂糖。每个ECIS的/出租车电极的8个独立的室组成。各室共用一个大电极（一），并包含一个单独的目标电极（B）。每个分庭也有2金圆（C），用于切割时用的套管井在琼脂糖为指导。黄金广场（D）的接触焊盘连接到POGO引脚，以适用于交流电流的电极。个人目标的电极是在电路中的每个较大的反电极（E）。 <img alt="图3" src="/files/ftp_upload/3840/3840fig3.jpg" /> 图3。两口井都切成室每个琼脂糖充满。当趋化以及被添加到一个创造最高最接近的趋化因子的趋化浓度梯度，在琼脂糖扩散。旅游下方琼脂糖对高浓度的趋化细胞，可以通过在目标电极。如细胞跨越的目标电极，阻力增加ECIS的1600R软件记录。 <img alt="图4" src="/files/ftp_upload/3840/3840fig4.jpg" /> 图4。两个14计成有机玻璃套管插入。一）一个5/64"钻头是用来锐化导管尖端，B）两孔必须钻成有机玻璃，以适应14计套管，根据布局的射门。c）用钻床，以确保孔垂直的pleixglass表面D）两个削尖的插管插入6.3"有机玻璃，相隔2毫米（从内缘计算）。这些削尖套管仔细插入琼脂糖削减井。 <img alt="图5" src="/files/ftp_upload/3840/3840fig5.jpg" /> 图5。 ECIS的/出租车室的黄金点对准套管。要削减井，的削尖套管必须ECIS的/出租车室的底部目标电极两侧的黄金点对齐。的套管应垂直插入，没有任何横向运动，并以同样的方式拆除。

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大卫克内希特和迈克尔Lynes的ECIS的/出租车的技术，作为被许可康涅狄格大学应用生物物理学，公司颁发的专利

ECIS的检测/出租车新颖等特点，包括其自动化实时数据的收集细胞以趋化因子的反应能力。虽然这项技术的最普遍的应用是测量细胞的反应，个别趋化梯度，或趋激动剂和拮抗剂的混合物组成的梯度，ECIS的/出租车方法的是服从这些配置的变化，这可能是非常有益的评估细胞的反应。有很好的证据，重叠或连续梯度可以影响细胞的行为，以新颖的方式。此外，它很可能是这些更为复杂的渐变是?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> ECIS的Zθ </td><td>应用生物物理学</td><td></td><td> <a href="http://www.biophysics.com/prodducts_Ecisz0.php">http://www.biophysics.com/prodducts_Ecisz0.php</a> </td></tr><tr><td> ECIS的电极阵列</td><td>应用生物物理学</td><td> 8W趋</td><td> <a href="http://www.biophysics.com/cultureware.php">http://www.biophysics.com/cultureware.php</a> </td></tr><tr><td> sea​​kem GTG琼脂糖</td><td> BioWhittaker分子应用</td><td> 50070 </td><td></td></tr><tr><td>培养液</td><td> cellgro </td><td> 10-040 </td><td></td></tr><tr><td>胎牛血清胎牛血清白蛋白</td><td> Thermo Scientific的</td><td> SH300703 </td><td></td></tr><tr><td> PEnicillin /链霉素</td><td> MP的Biomedicals </td><td> 1670049 </td><td>青霉素5000国际单位/毫升，链霉素5毫克/毫升</td></tr><tr><td> HEPES缓冲</td><td> MP的Biomedicals </td><td> 1688449 </td><td> 1M的解决方案，细胞培养级</td></tr><tr><td> 14号不锈钢套管（2）4英寸</td><td>一般实验室供应</td><td> 5-8365-1 </td><td>钝点</td></tr></tbody></table>

measurement of cellular chemotaxis with ecis/taxis

在回应外界刺激细胞运动是许多细胞过程，包括伤口愈合，炎症和感染的反应的基础。一种常见的方法来衡量趋Boyden小室检测，其中由多孔膜分离细胞和细胞趋化。通过向趋化膜作为细胞迁移，他们坚持膜，或下降到底层媒体的底面，并随后染色和视觉计算1。在此方法中，细胞暴露在一个陡峭的和短暂的趋化因子梯度，这被认为是2组织中发现的梯度差表示。 另一个检测系统，琼脂糖下的趋化检测，3，4项措施，各地根据琼脂糖凝胶层形成薄薄的水膜，在固体基质细胞运动。梯度在琼脂糖开发的是浅层的，被认为是一个应用程序ropriate代表性的自然发生的梯度。可行驶距离的显微成像评价趋。 Boyden小室法和下琼脂糖检测通常配置为端点检测。 ECIS的/出租车自动化系统相结合的电动细胞基质阻抗遥感（ECIS）的5，6下琼脂糖方法。在这个实验中，目标电极位于8室。一个大型的反电极贯穿每8室（ 图2）。每个室充满琼脂糖和两个小口井是在琼脂糖上削减目标电极的两侧。一个也充满了测试细胞的人口，而其他持有扩散的趋化因子的来源（ 图3）。通过系统的电流可以被用来确定在发生细胞越过目标电极的电阻变化。对靶细胞的焊条é增加阻力系统6。此外，在抵抗快速波动代表在细胞与电极表面相互作用的变化和正在进行的细胞形状变化的指示。 ECIS的/出租车系统可以在多长时间实时测量细胞群体的运动，但也很敏感，足以探测到一个单细胞，在到达目标电极。 被称为粘菌discoidium迁移叶酸梯度7，8 ECIS的/出租车9可以准确测量其趋化反应的存在。白细胞趋化反应到SDF1α趋拮抗剂也被ECIS的10，11 /出租车测量。一个白细胞反应SDF1α的例子如图1所示。

Watch this Scientific Journal Video about Measurement of Cellular Chemotaxis with ECIS/Taxis at JoVE.com

Measurement of Cellular Chemotaxis with ECIS/Taxis

企业家信心指数/出租车系统是一个自动化的，实时的检测，测量细胞趋化。在这个实验中，细胞的琼脂糖层到达目标电极下方移动。细胞运动耐交流电流0开始测量。

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

measurement-of-cellular-chemotaxis-with-ecistaxis

Research

JoVE Journal

Biology

14.7K 次观看.  University of Connecticut. 企业家信心指数/出租车系统是一个自动化的，实时的检测，测量细胞趋化。在这个实验中，细胞的琼脂糖层到达目标电极下方移动。细胞运动耐交流电流0开始测量。

视频: Measurement of Cellular Chemotaxis with ECIS/Taxis

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

使用微流体的细菌的趋化作用的研究 - 访

科研

生物学

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

膜片吸液管和夏普电极与一个可编程的拖轮

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

在学前儿童Neuromagnetic的脑功能自定义尺寸的MEG测量

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...