这项工作是支持由美国国立卫生/国立精神卫生研究所（NIH /镍氢电池）研究院授予＃1R21MH085267。

第1部分：准备多室模具主神经元共培养设备制造<ol><li>制备聚甲基丙烯酸甲酯主的第一步是使3.5毫米深的舱室。 SOMA舱和六个轴突/神经胶质细胞车厢定义有机玻璃块使用一个微型铣床（MDX - 40，罗兰或任何其他数控铣床）。聚甲基丙烯酸甲酯的主人是那么超声异丙醇10分钟，以清除杂物。 </li><li> 2.5微米高脊线结构的数组是一个标准的光刻工艺50.8毫米x 50.8毫米，湿蚀刻玻璃载玻片上图案。 首先，脊结构的数组与正光阻S1818清洗玻璃基板使用光刻图案。 S1818是旋涂在基板上（4000转），软在110 ° C为5分钟，然后暴露在紫外光灯（84兆焦耳/厘米2）使用光刻MF319光刻胶发展40秒。下一步，光致抗蚀剂图案的玻璃基板是沉浸在缓冲氧化层蚀刻（BOE）为6-7分钟，获得一个2.5微米高脊结构在室温下。最后，除去丙酮和异丙醇的光阻层，其次是在去离子（DI）水彻底​​冲洗。 </li><li>对PMMA主转让其模式与微脊结构的玻璃基板热压花。 首先，PMMA模具师傅和玻璃基板手动对齐和放置一个热压。然后，将温度提高到115 ° C。一旦达到所需的温度，适用于0.5吨的重量是5分钟。 5分钟后，温度被冷却到40 ° C和压力释放。聚甲基丙烯酸甲酯主，然后切成50.8毫米× 50.8毫米大小的块。 </li><li> PDMS预聚物（Sylgard 184）混合​​与固化剂的比例在10：按重量1。 </li><li>聚甲基丙烯酸甲酯的主人是放在一个木桶和PDMS的混合物浇聚甲基丙烯酸甲酯主上编造的PDMS主。然后放置在连接到真空泵的干燥器。 PDMS的倒在聚甲基丙烯酸甲酯主的顶部，然后是15分钟脱气，以消除在PDMS搅拌过程中产生的气泡。 </li><li>当所有的泡沫都将被删除，PDMS的一个拉平85 ° C烘箱内为一小时聚合。 </li><li>一旦PDMS完全治愈，PDMS的主剥下从PMMA主置于干燥器2-3滴的三氯内，15分钟的蒸汽大衣的PDMS主表面吸尘。三氯氢硅涂层，有利于最终PDMS的神经元共培养，从PDMS主设备释放。是简单地冲洗后的PDMS主涂层，异丙醇去除过量的三氯氢硅，并与 N 2气体干燥。 </li></ol>第2部分：神经元的文化设备复制<ol><li> PDMS预聚物（Sylgard 184）混合​​与固化剂的比例在10：按重量1。 </li><li> PDMS的混合物倒入的PDMS主上，并连接到一个15分钟的真空泵的干燥器内脱气。这一点很重要，不要倒太多的PDMS，使3.5毫米高的PDMS主结构是不完全沉浸。 </li><li>当所有的泡沫都将被删除，PDMS的一个拉平85 ° C烘箱内为一小时聚合。 </li><li>硅橡胶是从烤箱中取出一小时后，在室温下冷却下来。然后，PDMS的神经元的文化设备轻轻揭下从PDMS主。剥落的PDMS设备包装用封口膜，以保护他们免受灰尘或杂物。 </li><li> PDMS的设备，用封口膜包裹，切成各个部分使用一台钻床配备一个刀片。 </li></ol>第3部分：设备组装<ol><li> PDMS的神经元培养装置被放置在等离子体清洁和空气等离子暴露90秒钟，以使表面亲水性的PDMS设备。此等离子体处理将有利于文化传媒充满- D -聚赖氨酸涂层基板组装后的微通道。 </li><li>等离子体处理的设备，然后在70％的乙醇浸泡30分钟进行杀菌和消毒的生物引擎盖内移动。 </li><li>消毒设备是从70％乙醇和微孔过滤N 2气体的干燥。适用于6孔培养板和温和的压力，以确保基板和设备之间的密封，干燥设备被放置在聚- D -赖氨酸涂层的聚苯乙烯顶部。 </li><li>文化传媒，然后填充到SOMA舱，填补了六个轴突/神经胶质细胞车厢。最好是给1-2分钟灌装前轴突/神经胶质细胞后加入培养基SOMA车厢，以确保无气泡内的微被困车厢的暂停。 </li><li>充满文化传媒的设备内的37 ° C培养箱中过夜冲洗任何可能的碎片或有毒残留物孵育。 </li></ol>第4部分：细胞装载和神经元，少突胶质细胞共培养<ol><li>在第一天的细胞培养，原发性脑的神经细胞从胚胎一天16-18大鼠加载到SOMA车厢的500个细胞/平方毫米的磁录密度。首先，SOMA车厢和轴突/神经胶质细胞车厢内的文化传媒细胞加载之前使用吸管吸气。然后，38500细胞，在40μL培养基稀释，被加载到微进气口周围的SOMA车厢。重要的是不要触摸的PDMS设备，同时加载的细胞，因为它可以打破设备和基板之间的密封。 </li><li>孵育30分钟内的37℃培养箱中培养的细胞加载装置，以提高细胞对基材的附着力。孵化后，所有的车厢都充满了文化传媒。 </li><li>细胞培养于37 ° C和饱和湿度5％CO2培养箱中只有一半是文化传媒改为每3-4天。 </li><li>经过14天在体外培养的神经元，少突胶质细胞轴突/共培养神经胶质细胞车厢。从1-2日龄Sprague - Dawley大鼠大脑半球的解剖，少突胶质细胞，被添加到每个轴突/神经胶质细胞的车厢，在1000个细胞/平方毫米的面积密度。装载细胞之前，约60％的车厢内的文化传媒与吸管吸气。必须注意不要触摸底部基板，因为它可能会损坏轴突在基板上形成的网络。然后，6700少突胶质细胞，在5μL培养基稀释，添加到轴突/神经胶质细胞车厢。细胞孵育1小时，车厢内都充满了文化传媒。 </li><li>细胞培养在37℃饱和湿度5％的CO 2培养箱中，只有一半是文化传媒改变每隔3-4天，两个星期中的C。 </li></ol>第5部分：代表性的成果： 当实验进行正确，加载到SOMA车厢神经细胞定位密切微进水口和轴突层开始后1周的文化，形成轴突/神经胶质细胞车厢内。登录SOMA车厢内的神经细胞聚集，可以是一个迹象，细胞并不健康。当装载少突胶质细胞神经​​元文化的两个星期后应覆盖一层致密的轴突和少突胶质细胞，轴突/神经胶质细胞车厢应轴突层的顶部装上。 <img src="/files/ftp_upload/1399/1399fig1tmb.jpg" border="0" alt="图1" /> 图1。示意图插图的高通量微流体多室中枢神经系统神经元共培养平台。剖视图显示从神经元胞体和树突，以及流体分钟流体水平的差异车厢之间的隔离的轴突隔离。请<a href="https://www.jove.com/files/ftp_upload/1399/1399fig1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1399/1399fig2tmb.jpg" border="0" alt="图2" /> 图2多舱的制造和装配步骤的PDMS微流体合作文化设备。每个设备都适合到一个传统的6孔聚苯乙烯细胞培养板。请<a href="https://www.jove.com/files/ftp_upload/1399/1399fig2.jpg">点击这里</a>看大图图2。

<ol>
	<li>Sherman, D. L., Brophy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+axon+ensheathment+and+myelin+growth.">Mechanisms of axon ensheathment and myelin growth.</a> Nature Reviews. 6, 683-690 (2005).</li><li>Xia, Y., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography.">Soft Lithography.</a> Angew. Chem. 37, 550-575 (1998).</li><li>Taylor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature Methods. 2, 599-605 (2005).</li><li>Li, J., Baud, O., Vartanian, T., Volpe, J. J., Rosenberg, P. A., A, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxynitrite+generated+by+inducible+nitric+oxide+synthase+and+NADPH+oxidase+mediates+microglial+toxicity+to+oligodendrocytes.">Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes.</a> Proceedings of the National Academy of Sciences. 102, 9936-9941 (2005).</li></ol>

The authors have nothing to disclose.

我们已经开发出一个多车厢文化合作平台，为研究哺乳动物中枢神经系统轴突的神经胶质细胞相互作用。中枢神经系统轴突被成功分离，从神经细胞团体/树突和少突胶质细胞内的设备已成功合作培养。神经元密度可以是多种多样的应用程序，但浓度过低或过高，可导致细胞死亡。此外，重要的是改变培养基的只有一半，在基板上，使细胞或轴索层不被损坏。我们预计，该系统将会为研究中枢神经...

a multi-compartment cns neuron-glia co-culture microfluidic platform

我们目前在体外中枢神经系统轴突的神经胶质细胞相互作用的研究一种新型的神经元多室共培养微平台，能够进行高通量平行的6个独立的实验。我们开发了一种新的制造方法，以建立具有微观和宏观尺度的结构，通过一个单一的软光刻工艺相同设备内的微流体装置，使大规模制造，具有良好的重复性。 多室微合作文化的平台是一个神经元轴突/神经胶质细胞和少突胶质细胞（OLS）6车厢SOMA车厢组成。 SOMA舱和轴突/神经胶质细胞车厢连接阵列轴突指导微函数作为物理屏障只在SOMA车厢中的神经元胞体，同时允许轴突轴突/神经胶质细胞车厢长成。装入轴突/神经胶质细胞车厢的OLS可以交互只与但不与神经元的胞体或树突的轴突，使局部轴突的神经胶质细胞相互作用的研究。微也使车厢之间的流体隔离，允许6个独立的实验，以一个单一的设备上进行更高的吞吐量。 使用聚二甲基硅氧烷（PDMS）的软光刻技术在生物医学常用的微型器件。在这些设备上的水库，通常定义的手动冲床。虽然简单，穷人的路线和时间费时的过程，使得这个过程中不适合时，大量的水库已被反复创建。新开发的方法并不需要手动冲压水库，克服这些局限性。首先，7个水库（深：3.5毫米），上一聚（甲基丙烯酸甲酯）（PMMA）使用的微型铣床块。然后，脊微观结构的阵列，在玻璃基板上制造，对有机玻璃块定义胞体和轴突/神经胶质细胞车厢连接的微热压印。这个过程导致了宏观尺度的水库（3.5毫米）和微观尺度通道（2.5微米），在一个单一的聚甲基丙烯酸甲酯主不谋而合。一个PDMS的副本作为一个模具师傅用软光刻技术和最终的PDMS设备是从这个主复制。 E16 - 18只大鼠的初级神经元被装到SOMA室和培养两个星期。细胞培养一周后，轴突越过微，形成轴突轴突/神经胶质细胞车厢内唯一的网络层。整个六轴突/ P1 - 2只大鼠神经胶质细胞车厢和OLS均匀轴突增长在14 天的体外补充轴突/神经胶质细胞车厢的共同培养。

Watch this Scientific Journal Video about A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform at JoVE.com

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

我们开发了一种新型的多舱的神经元共培养的微平台，在体外培养的中枢神经系统的轴突神经胶质细胞相互作用的研究。该平台能够进行多达6个独立并行实验，并采用新开发的宏观/微观混合制备方法。

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 ...

a-multi-compartment-cns-neuron-glia-co-culture-microfluidic-platform

Research

JoVE Journal

Biology

11.9K 次观看.  Texas A&M University (TAMU). 我们开发了一种新型的多舱的神经元共培养的微平台，在体外培养的中枢神经系统的轴突神经胶质细胞相互作用的研究。该平台能够进行多达6个独立并行实验，并采用新开发的宏观/微观混合制备方法。

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

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 ...

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

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 ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

根据流量的血小板粘附和聚集，使用微流控流细胞

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

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 ...

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.

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 ...

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

修改ES / OP9共培养协议，提供增强造血后代的表征

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...