这项工作是支持部分由美国国家科学基金会（CBET 0846453）。

1。制备硅的主人，使用标准的SU - 8光刻1（在这个视频没有显示）。 <ol><li>使用标准的SU - 8光刻方法，编造包含微流体网络和趋化室的PDMS模具的苏- 8“主”（SU - 8 2025年，MicroChem，牛顿，MA）的创建。这种掌握模具，可以制作任何微细加工设施（例如，斯坦福大学的微流体铸造<a href="http://thebigone.stanford.edu/foundry/">;</a> http://thebigone.stanford.edu/foundry/） ​​。 </li><li>的PDMS复制之前，公开的SU - 8的主蒸汽fluorosilane在连接到真空源，以方便从主PDMS容易释放干燥器中添加fluorosilane下降到纸巾，并申请一分钟的真空。拆下真空，并允许30分钟fluorosilane沉积。存放在密闭容器中以备将来使用SU - 8的主。 </li></ol> 2。翻版硅橡胶成型，从SU - 8主：流体层和控制层是由硅橡胶成型的SU - 8主副本。 <ol><li> 10:1 WT混合的PDMS预聚物和交联剂。比和德加为1小时（或直到气泡从PDMS的混合物中删除）dessicator。 </li><li>放置在培养皿SU - 8的掌握和仔细的SU - 8的掌握上的PDMS混合物倒到所需的厚度。 </li><li>热的培养皿中含有的PDMS和在80的SU - 8母模° C至两小时治愈的PDMS。 </li><li>取出固化硅橡胶覆盖从烤盘SU - 8的掌握和剥离的PDMS模具从SU - 8主。所需的结构将被嵌入在PDMS模具。 </li><li>管梯度发生器和细胞的入口，使用20号钝针头的PDMS模具打孔。 </li></ol> 3。 PDMS的设备的粘接： <ol><li>清洁玻璃显微镜玻片与异丙醇和干燥的氮气或空气流。 </li><li>公开的PDMS和载玻片氧等离子体在等离子体ASHER。 </li><li>带入接触载玻片的PDMS模具。联系幻灯片和PDMS模具加热至65 ° C，持续15分钟。 </li></ol> 4。组装的PDMS设备<ol><li>使用刀片，切管在45 °角设备所需的正确的长度。 </li><li>管子的一端插入到设备使用镊子（例如，插座）砸出的洞。 </li><li>使用产钳，油管的另一端插入一个生硬的30号针头。 </li><li>所有剩余的孔，重复步骤4.2。 </li><li>在3ml的注射器收集的趋化缓冲液（CB）1ML，照顾，从注射器中删除的气泡。 </li><li>插座管的一端固定一针枢纽。 </li><li> CB针枢纽，挖掘针枢纽，以消除任何气泡。 </li><li>推注射器的尖端的小CB出来，没有诱捕空气连接3ml的注射器针头枢纽。 </li><li>推动通过设备CB与CB填充，直到所有剩余的针枢纽。这应该删除的大部分气泡。 </li><li>填写两个500μL注射器与CB包含chemoeffector正在测试适当的浓度。消除气泡的形成，推动注射器内容的一个小水滴接触的液体滴在针枢纽和附加。 </li><li>同样，连接注射器，含CB细菌入口。当细菌进入设备，这将被删除。 </li></ol> 5。高运动型大肠杆菌的生长大肠杆菌 <ol><li>种植过夜培养的大肠杆菌大肠杆菌 RP437与绿色荧光蛋白表达质粒在20毫升蛋白胨肉汤（TB）在32 ° C，用颤抖的。添加适当的抗生素浓度以维持质粒。在我们的协议中，我们使用一个基于荧光设备的细菌检测低拷贝的质粒pCM182。为E.大肠杆菌日益增长的文化，在32 ° C，这样高蠕动，蠕动在较高的温度低。 </li><li>过夜培养，接种在250 mL锥形瓶中，在600nm处的光密度为0.05〜20 mL培养的结核病。文化成长与晃动在32 ° C。 </li><li>收获低速离心5分钟XG 400外径600的0.45〜0.35的细胞。 </li><li> 〜0.35 OD在CB 600预计在通道中间的浓度含的chemoeffector重悬细胞。 </li><li> RFP标记的死细胞，活细胞表达GFP的加一个外径600〜0.35。死细胞（红色）被用来作为内部控制，以确保任何迁移是不是由于流动效应。 </li></ol> 6。监测的趋化作用<ol><li>取出的细胞进针的CB枢纽。笔芯与细胞悬液。 </li><li>轻轻地填写与悬浮细胞50μL注射器。这一步需要做，慢慢鞭毛可以剪切，并会减少蠕动。 </li><li>将50μL注射器步骤4.10所述的进气针枢纽。 </li><li>设备上配有一个高速摄像机进行图像采集，在荧光显微镜阶段的位置。放入注射泵的进气口注射器（无论是500μL，梯度注射器和50μL细胞注射器），并启动梯度形成和细胞通过油管流向流量等。 </li><li>一旦细胞进入趋化室，等待系统〜20分钟前成像稳定。 </li><li>收集沿腔的长度不同的位置（对应不同的曝光时间），绿色（居住）和红色荧光图像（死）。通常情况下，我们在每个位置3秒的间隔收集的100幅图像。 </li></ol> 7。数据分析：使用市面上任何图像分析程序（例如，ImageJ，Metamorph）或使用在Matlab编写的简单代码，可以执行下列步骤。 <ol><li>删除背景像素的图像（即设定的阈值的像素强度，并删除所有具有强度低于阈值的像素）。这在降低噪声的分析。 </li><li>使用的死细胞（红色荧光）的位置，以确定图像的中心。由于死细胞没有趋化，这代表细胞进入趋化室，将在没有任何迁移检测的位置。 </li><li>找到图像中的活细胞（绿色荧光）。 </li><li>分成渠道的形象，并确定每个通道中的活细胞的数量。在我们以前的工作，1050μm宽的趋化室被分为64个通道，每个〜16微米宽。 </li><li>重复步骤7.1 7.3的所有图像，所有图像的每个通道的总细胞计数相加。这使得在实验期间的每个通道检测细胞总数。 </li><li>计算的趋化分配系数（中共），它代表的迁移方向，如下：在高浓度（右）和低浓度（左）双方的死细胞位于每个单元分配一​​个倍数+1和-1分别。也就是说，一个细胞迁移渐变乘以1，而细胞向下移动渐变乘以-1。所有乘以值加起来和标准化检测细胞的总数。例如，中国共产党的价值为0.40表明，细菌总数增加了40％，比低浓度一侧（即chemoeffector是诱食，为更多的细菌检测移动渐变）高浓度侧。 </li><li>计算的趋化迁移系数（CMC），重量细胞迁移距​​离的旅行，如下：重量细胞的每个通道中的一个因素，是细胞迁移的距离成正比的计数。一个细胞移动到最远的高浓度的位置（64通道）是一个加权系数+1，而移动一半浓度较高侧给出了0.5的加权系数，并移动到最远的一个细胞低浓度的立场是加权-1。总结所有的加权细胞计数和正常化的细胞数量产生管委会。 </li></ol>代表性的结果3,4： 我们已经使用了微流体装置（ 图1A）这里描述的调查大肠杆菌趋化大肠杆菌的引诱（天门冬氨酸，autoinducer - 2）和驱虫（NISO 4，吲哚）3,4梯度。典型的趋化应变5 E.大肠杆菌 RP437表达GFP一起使用，与卡那霉素死亡E.大肠杆菌 TG1中表达红色荧光蛋白监测流动效应。在μFlow设备中形成浓度梯度， 如图1b所示。所以，有没有通过它的流量和一个稳定的梯度0 100毫微克/毫升fluoresscein，在设备，单元入口加盖。器件上的像素点的亮度来确定的荧光素浓度剖面。数据显示，细菌遇到的线性渐变，当他们进入趋化室。图2A和 B 显示 E.荧光图像大肠杆菌 RP437天门冬氨酸（0 100微米）和NISO 4（0 225微米）的梯度迁移。所观察到的反应，这些规范的诱食和驱蚊预测图3A显示E.量化分配大肠杆菌 RP437 在浓度梯度的情况下（即，空，天门冬氨酸的梯度），而图3B和C，天门冬氨酸和NISO 4梯度分布。这些反应的特异性（即，在移民的偏见）是显而易见的，作为一个E。 大肠杆菌 RP437Δ 焦油应变（即应变缺乏在大肠杆菌中的焦油化学感受器是感天门冬氨酸和镍），并不能说明在迁移中存在的天门冬氨酸或NISO 4的浓度梯度的偏见。在空间的分布状况的明显趋势，也与计算的中国共产党和中央军委值一致。 E 的迁移，中国共产党重视大肠杆菌 RP437在4天门冬氨酸或NISO的梯度，分别为0.33和-0.33。相应的CMC值分别为0.13和-0.14。相比之下，中国共产党和CMC值的大肠杆菌大肠杆菌 RP437Δ 焦油的天门冬氨酸或NISO 4的应变梯度可以忽略不计。此外，中共中央和中央军委的天门冬氨酸的空梯度，分别为0.03和0.02，并注明缺乏迁移的偏差，在渐变的情况下。 <img src="/files/ftp_upload/1779/1779fig1a.jpg" alt="图1a" /> <img src="/files/ftp_upload/1779/1779fig1b.jpg" alt="图1b" /> 图1。设备原理图和浓度梯度 。 （一）微流体的趋化设备的示意图。该装置由一个渐变混合模块（20 × 100 × 18750微米）和趋化观察模块（20 × 1050 x 11500微米）。梯度进入设备的两个渠道的宽度是500微米，细菌入口的宽度为50μm。插图描绘了一个渐变的信号分子（灰色）和细菌在它迁移。活的细菌被描述为实心椭圆形，而死亡的细菌是开放式的椭圆形所示。 （B）在0和100纳克/毫升荧光之间的浓度梯度生成设备和成像用荧光显微镜。被收购后的30分钟的荧光图像和图像分析量化。 <img src="/files/ftp_upload/1779/1779fig2.jpg" alt="图2" /> 图2。趋化E。大肠杆菌 RP437在微流体装置。 大肠杆菌 RP437暴露的梯度（一）0-100微米L -天门冬或（B）0-225微米NISO 4设备和活的细菌迁移（绿色）对L -天门冬或远离镍成像为30分钟，每2.5秒。死菌（红色）担任控制设备中的流动效应。使用一个内部开发的分析program3的图像进行了量化。数据显示，有代表性的3次独立实验的伪彩色图像。 <img src="/files/ftp_upload/1779/1779fig3a.jpg" alt="图3a" /> <img src="/files/ftp_upload/1779/1779fig3b.jpg" alt="图3b" /> <img src="/files/ftp_upload/1779/1779fig3c.jpg" alt="图3c" /> 图3：迁移型材量化到一个规范的引诱剂和驱虫剂。 在微流体装置的空间分布RP437在回应（一）统一的100微米L -天门冬（即无梯度），（二）0 100天门冬氨酸微米梯度，和（C）0 225 NISO 4微米梯度。 RP437细胞的分布是死细胞的分布是实线所示，虚线所示， 和 RP437 EDA +Δ焦油细胞的分布是一条虚线所示。数据显示，平均为3次独立实验。

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Hansen, M. C., Palmer, R. J. J., Udsen, C., White, D. C., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+GFP+fluorescence+in+cells+of+Streptococcus+gordonii+under+conditions+of+low+pH+and+low+oxygen+concentration.">Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration.</a> Microbiol. 147, 1383-1391 (2001).</li><li>Englert, D. L., Manson, M. D., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Flow-Based+Microfluidic+Device+for+Quantifying+Bacterial+Chemotaxis+in+Stable,+Competing+Gradients.">A Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients.</a> Appl Environ Microbiol. 75, 4557-4564 (2009).</li><li>Englert, D. L., Jayaraman, A., Manson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidic techniques for the analysis of bacterial chemotaxis. , (2009).</li><li>Mao, H., Cremer, P. S., &amp;amp, M. a. n. s. 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=A+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> PNAS. 100 (9), 5449-5454 (2003).</li></ol>

在毛泽东等人 5所描述的趋化分配系数（CPC）和趋化迁移系数（CMC），可以计算出。如果一个细胞上检测到高浓度侧，它是一个价值1，而在低浓度侧检测的单元格值-1。值总结和细胞总数除以产生中国共产党。中国共产党的符号（正或负）给出的迁移方向（朝向或远离信号）。虽然中国共产党表示是否细胞作为一种化学引诱或驱蚊剂，它没有量化的趋化程度。对于这一点，我们除?...

a microfluidic device for quantifying bacterial chemotaxis in stable concentration gradients

趋使细菌方法诱食化学品的来源，或避免驱蚊化学品的来源。细菌不断地监视当前浓度的浓度进行比较具体chemoeffectors的浓度检测早几秒钟。这种比较，决定运动的净方向。虽然多个，竞争梯度往往共存于大自然，调查细菌的趋化作用的常规方法是最理想量化引诱剂和驱虫的浓度梯度迁移。在这里，我们描述了一个微流体的趋化模型，提出chemoeffectors精确和稳定的浓度梯度细菌和定量调查应用梯度的响应的发展。该装置是在任何所需的绝对浓度和梯度强度，浓度梯度的多功能，可以很容易扩散混合产生。该设备是证明使用的响应<em&gt;大肠埃希氏菌</em&gt; RP437氨基酸和镍离子的梯度。

Watch this Scientific Journal Video about A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients at JoVE.com

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

本协议描述了一个微流体装置的发展，为细菌的趋化调查chemoeffectors稳定的浓度梯度。

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 ...

a-microfluidic-device-for-quantifying-bacterial-chemotaxis-stable

Research

JoVE Journal

Biology

12.1K 次观看.  Texas A&M University. 本协议描述了一个微流体装置的发展，为细菌的趋化调查chemoeffectors稳定的浓度梯度。

视频: A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

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

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 ...

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

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 ...

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

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

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

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 ...