这项工作得到了国际奖学金AAUW Adeola Adewola，美国国立卫生研究院/何塞Oberholzer NCRR（U42RR023245），和芝加哥糖尿病项目。

A. 3层的微流体perifusion装置微细底部井主协议（150微米深水井） <ol><li>如果需要使用刀片清洁晶圆。与丙酮，甲醇，异丙醇清洁。执行为30秒50瓦的血浆治疗</li><li>自旋SU8 - 100 @ 2000转。 [步骤1：500 RPM，10秒，100转/秒，第2步：2000转，30秒，300转/ S]。 [注：不与晶圆镊子后纺SU8] </li><li>软烤晶圆在65 ° C为20分钟和95 ° C为50分钟。 </li><li>晶圆被暴露在紫外线下，通过所需的面具。剂量为150微米的高度是650兆焦耳/ 厘米 2 。 </li><li>暴露后烤晶圆65 ° C为1分，在95 ° C为12分钟。 </li><li>发展在15分钟的SU8开发晶圆。 </li></ol>微通道主协议（500微米深井​​） <ol><li>如果需要使用刀片清洁晶圆。与丙酮，甲醇，异丙醇清洁。执行为30秒50瓦的血浆治疗</li><li>自旋SU8 - 2150 @ 1000转。 [步骤1：500 RPM，10秒，100转/秒，第2步：1000转，30秒，300转/ S]。 </li><li>软烤晶圆在65℃，15分钟和95 ° C时2小时30分钟。 </li><li>晶圆被暴露在紫外线下，通过所需的面具。曝光剂量为650微米的高度是685兆焦耳/厘米2。 </li><li>暴露后烤晶圆65 ° C为5分钟，在95 ° C为35分钟。 </li><li>发展在20-30分钟的SU8开发晶圆。 </li></ol> PDMS的解决方案的准备<ol><li>聚二甲基硅氧烷（PDMS）的解决方案是准备彻底搅拌10部分有机硅弹性体与1固化剂的标准Sylgard 184套件的一部分。 </li><li>使用真空干燥器，在PDMS的解决方案，在搅拌过程中产生的气泡被删除。 </li><li>无气泡的PDMS的解决方案是慢慢地配发到SU8船长和一个空的第三层的Petri菜。 </li><li>热板的温度设定到75 ° C，2小时，在此温度下固化的PDMS </li><li>入口，出口和汇率井出使用适当大小的打孔器打孔。 </li><li>这些层粘合在一起，在玻片上，用一个手持血浆设备（尺寸0.1毫米）。 </li></ol> B.实验装置<ol><li>通过微设备的流量70％乙醇消毒。流量DI水，洗出的乙醇。灌注50毫升0.5％BSA，通过设备，以防止胰岛素的非特异性吸附微墙壁。 </li><li>葡萄糖坡道梯度和LabVIEW软件，通信用的注射器泵测试，以确保梯度稳定所产生的其他相关梯度。 </li><li> 25-30小鼠胰岛共孵育5微米Fura-2/AM（钙指标，分子探针，CA），在37℃30分钟的2.5微米罗丹明123（Rh123的，线粒体的潜力指标，Sigma公司，MO）克雷布斯的林格氏缓冲液（KRB）含有2毫米葡萄糖</li><li>小鼠胰岛仔细吸管到微流体perifusion装置，通过进气口。 </li><li>微流体网络，然后通过注射泵使用聚乙烯管和一个Y型连接器和馏分收集器的电源插座连接的入口设置。该设备坐落在加热阶段（37℃），在显微镜的进油管上烤盘加热室和解决方案的温度保持在37 ° C。 </li><li>安装后，立即小鼠胰岛perifused KRB包含10分钟，然后葡萄糖坡道为25分钟（2MM 25MM）2毫米葡萄糖。 </li><li>定时图像收集和分析SimplePCI软件每15秒。 perifusate是收集每分钟使用馏分收集，分析胰岛素的分泌，使用试剂盒。 </li></ol>代表性的成果小鼠胰岛perifused 2-25葡萄糖的线性渐变。正如图1所示，钙离子内流和胰岛素分泌被触发后约13分钟的perifusion，对应到6毫米葡萄糖。线粒体电位的变化早些时候预计，在约11分钟。这一数据表明，使用这种微流体网络的优势，胰岛生理特征。 <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="图1" /> 图1。胰岛细胞刺激的生理反应。

<ol>
	<li>Mohammed, J. S., Wang, Y., Harvat, T. A., Oberholzer, J., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+multimodal+characterization+of+pancreatic+islets.">Microfluidic device for multimodal characterization of pancreatic islets.</a> Lab Chip. 9, 97-106 (2009).</li></ol>

传统的胰岛perifusion系统（宏观和微观）有一定的限制设置和设计，技术要求高，难度在系统中创建的用户指定的化学梯度的复杂。这里描述的微流体perifusion系统克服了这些限制，简单的几何设计和制造。更重要的是，这个系统可以集成与荧光成像方法，提供一个独特的工具来研究胰岛生理。证明该系统具有高信噪比和这些荧光信号的时空分辨率。目前的原型设备，也可以在一个芯片上容纳多个pe...

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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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<tr>
<td>60ml Syringes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fura-2 fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine123 Fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>30" Silicone tubings</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16 x 1/8in</td>
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<tr>
<td>1.5ml Eppendorf tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Y-connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D; &amp; 4mm</td>
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<tr>
<td>Syringe connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>female luer plug 1/16&#x201D;</td>
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<tr>
<td>Straight connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
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<tr>
<td>Elbow connector</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
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<tr>
<td>Havard syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Perifusion device</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hot plate</td>
<td>&nbsp;</td>
<td>PMC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Thermometer</td>
<td>&nbsp;</td>
<td>Omega Engineering Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fraction collector</td>
<td>&nbsp;</td>
<td>Gibson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pippettor</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Inverted epiflorescence microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>IX71</td>
<td>&nbsp;</td>
<tr>
<td>Charge-coupled device</td>
<td>&nbsp;</td>
<td>QImaging</td>
<td>&nbsp;</td>
<td>Retiga-SRV, Fast 1394</td>
</tr>		</tbody>
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a multi-parametric islet perifusion system within a microfluidic perifusion device

一个微流体胰岛perifusion装置，开发了多个小岛和钙离子内流和线粒体的潜在变化的同步荧光成像的动态胰岛素分泌的评估。该装置由三个层次：第一层包含一个微型井（直径为500微米和150微米的深度），帮助固定的小岛，而暴露流量和胰岛暴露的表面积最大化的数组，第二层包含循环perifusion室（3毫米深，直径7毫米）;和第三层包含一个入口，混合通道，球迷前注入perifusion室（2毫米宽，19毫米的长度，高度在500微米）优化混合效率之前进入perifusion室。创造各种糖梯度，包括线性，钟形，方形，也可以在微流体perifusion网络，是证明。

Watch this Scientific Journal Video about A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device at JoVE.com

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

一个微流体胰岛perifusion装置，开发了多个小岛和钙离子内流和线粒体的潜在变化的同步荧光成像的动态胰岛素分泌的评估。

一个多参数的胰岛内的微流控Perifusion装置Perifusion系统

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

a-multi-parametric-islet-perifusion-system-within-microfluidic

Research

JoVE Journal

Biology

11.8K 次观看.  University of Illinois, Chicago. 一个微流体胰岛perifusion装置，开发了多个小岛和钙离子内流和线粒体的潜在变化的同步荧光成像的动态胰岛素分泌的评估。

视频: A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

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 ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

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.

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Subtitles

一个多参数的胰岛内的微流控Perifusion装置Perifusion系统