我们要感谢让我们电影的一部分，这在洁净室设施的视频，在麻省理工学院微系统技术实验室。

制备 1。创建遮罩<blockquote>使用CAD软件，设计了一个透明度高清晰度印刷的渠道。这将是“面具”。 </blockquote>在洁净室： 2。清洁和烤晶圆首先，喷出的晶圆与丙酮，然后迅速与甲醇，然后用异丙醇。最后，用氮气干燥晶圆。 在5分钟的烤箱（130℃）烘烤晶圆。 3。涂层的晶圆晶圆放置在旋转涂布机的中心。倒入一瓶到晶圆光阻（SU - 8）。让我们的SU - 8流和放松〜10秒。打开自旋涂布机和斜加快从0到500 RPM超过5秒;保持在500转10秒;上升超过10秒，最终速度和最终速度保持30秒。最终的速度取决于针对性的涂层厚度，并用SU - 8。的细节，可以发现<a href="http://www.microchem.com/">在</a> http://www.microchem.com/ 4。软烤晶圆涂层后，烘烤在65 ° C，然后在95 ° C。烘烤时间随有针对性的厚度和光致抗蚀剂的类型。然后，让晶圆在室温下坐了至少5分钟。 5。曝光晶圆上的面具，暴露在SU - 8手册建议的时间晶圆紫外灯。 6。后曝光烤晶圆烘烤在65 ° C和95 ° C以下的SU - 8手册的指示。 7。发展晶圆获得的“主人”（模具） 准备充满开发甲基丙烯酸甲酯（PMMA）的烧杯。晶圆浸泡放入烧杯中，轻轻振荡烧杯，直到未曝光部分的光刻胶被冲走。 在我们的实验室： 8。准备PDMS和倒到晶圆在10:1的比例进入一个杯子，与固化剂混合的PDMS。搅拌混合均匀：这将产生大量的气泡，使混合物看起来不透明。倒在“大师”的混合物。 9。泡沫在真空室要除去气泡，放置的掌握和PDMS的混合物，覆盖到一个真空室，直到所有的气泡都没有了。 10。烘烤在烤箱 烘烤至少12小时在65 ° C至变硬的​​PDMS烤箱。 11。打孔剥离的PDMS从主渠道的进水口和网点打孔。 在洁净室（未显示） 12。等离子粘接通道是一个处理后的PDMS层和1分钟的等离子体与氧气的载玻片载玻片保税。 实验： EXP＃1：研究海洋微生物的趋化反应微观尺度的营养层 1）建立实验<ol><li>添加生物和基板玻璃注射器</li><li>放置到显微镜下阶段的微流体通道，并附加油管适当的入口和出口</li><li>连接管废物水库。确保管是完全浸没在流体中的浪费水库，以避免压力振荡</li><li>广场注射器注射泵上，并连接到阀门和管路</li><li>显微镜：光照条件下，放大倍数等</li><li>重点在适当的位置上通道</li><li>德泡沫通道使用较大的注射器充满人工海水</li><li>设置适当的流速注射泵。在这种情况下，2毫升/分钟，这相当于一个220微米s - 1时的平均流速通道</li></ol> 2）运行实验<ol><li>启动注射泵，以建立一个通道的营养梯度</li><li>一旦流量已趋于稳定，并已开发的营养带，停止注射泵的流量和从这点开始录制时间</li><li>营养乐队开始横向扩散</li><li>在固定的时间间隔，使用图像分析软件，记录帧序列，以创建“电影” </li><li>随后的两个帧之间的时间差的图像，使移动的物体是只有现在的可视化，让我们以区别于非移动的粒子和背景噪音的游动细胞歧视游泳生物体</li><li>以电影的细胞，以确定职位与参考通道的Position的营养补丁</li><li>在10-20分钟的定期记录影片，以分析生物体的立场和游泳模式</li><li>通过使用图像分析软件，叠加在不同的帧（分配到每个像素的像素记录在电影期间的最大光强）的生物体的位置，我们可以得到游泳细胞的运动轨迹信息</li></ol> EXP＃2：调查对海洋细菌的剪切效应，在一个vortexZ游泳<ol><li>使用不同的渠道几何，我们可以观察到的行为在不同的剪切速率在旋涡游泳的细菌</li></ol>

<ol>
	<li>Azam, F., Malfatti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+structuring+of+marine+ecosystems.">Microbial structuring of marine ecosystems.</a> Nature Reviews Microbiology. 5, 782-791 (2007).</li><li>Blackburn, N., Azam, F., Hagstrom, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+explicit+simulations+of+a+microbial+food+web.+.">Spatially explicit simulations of a microbial food web. .</a> Limnology and Oceanography. 42, 613-622 (1997).</li><li>Blackburn, N., Fenchel, T., Mitchell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nutrient+patches+in+plankton+habitats+shown+by+chemotactic+bacteria.">Microscale nutrient patches in plankton habitats shown by chemotactic bacteria.</a> Science. 282, 2254-2256 (1998).</li><li>Keymer, J. E., Galajda, P., Muldoon, C., Park, S., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+metapopulations+in+nanofabricated+landscapes.">Bacterial metapopulations in nanofabricated landscapes.</a> Proceedings of the National Academy of Science. 103, 17290-17295 (2006).</li><li>Mao, H., Cremer, P. S., 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+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> Proceedings of the National Academy of Science. 100, 5449-5454 (2003).</li><li>Marcos, R., Stocker, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+in+vortices:+a+microfluidic+setup.">Microorganisms in vortices: a microfluidic setup.</a> Limnology and Oceanography: Methods. 4, 392-398 (2006).</li><li>Park, S., Wolanin, P. M., Yuzbahyan, E. A., Lin, H., Darnton, N. C., Stock, J. B., Silberzan, P., Austin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+topology+on+bacterial+social+interaction.">Influence of topology on bacterial social interaction.</a> Proceedings of the National Academy of Sciences. 100, 13910-13915 (2003).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <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+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li></ol>

海洋微生物如何与当地的化学和物理环境相互作用的理解是势在必行的浮游微生物在海洋中的养分和碳循环（2007年阿扎姆和Malfatti）中的作用更加完整和准确的的感知。然而，由于许多重要的微生物的相互作用，哪些发生小尺度（毫米），技术上的限制，阻碍内异质景观生物物理化学，微生物行为的详细检查，预计要在海洋中游泳的微生物的经历。在微流体（怀特赛德斯等，2001）的最新进展使微生物生态学的详...

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<td>PDMS, Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
<td>Dow Corning, Midland, MI, USA</td>
<td>&nbsp;</td>
<td>http://www.ellsworth.com/sylgard.html</td>
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<td>SU8-2100</td>
<td>Photoresist</td>
<td>MicroChem, Newton, MA, USA</td>
<td>&nbsp;</td>
<td>www.microchem.com
</td>
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<td>Nikon Eclipse TE2000-E inverted microscope</td>
<td>Microscope</td>
<td>Nikon, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PEEK tubing (0.762 mm ID, 1.59 mm OD)</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>www.upchurch.com</td>
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<td>Syringes (Luer-Lok Tip)</td>
<td>Tool</td>
<td>BD, Franklin Lakes, NJ, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fitting Part P-704-01</td>
<td>Tool</td>
<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
<td>&nbsp;</td>
<td>To connect tubing to Luer-Lok Tip Syringes</td>
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<td>Syringe Pump (PHD 2000 Programmable)</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CCD Camera (PCO 1600)</td>
<td>Equipment</td>
<td>Cooke, Romulus, MI, USA</td>
<td>&nbsp;</td>
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chemotactic response of marine micro-organisms to micro-scale nutrient layers

浮游的微生物可以利用微型的资源的修补程序，在何种程度上，将有相当大的影响，为海洋trophodynamics和生物地球化学通量。然而，要在海洋营养补丁优势，游泳微生物必须克服身体力量的影响，包括分子扩散和湍流剪，这将限制的修补程序的可用性和细菌的能力找到他们。直到最近，方法上的限制，排除内修修补补的栖息地和现实的小规模流动条件下的微生物行为的直接考试。因此，我们目前的知识关于在海洋中的微生物的行为已经从理论预测采购。为了获得我们已申请了软光刻制造技术，开发微流体装置，这是我们用来创建（一）微型尺寸和扩散的特点有关海洋过程的营养补丁及（ii）微尺度海洋微生物觅食行为的新信息与剪切率对应预计在海洋的旋涡。这些微流体装置允许在异构和动态的海景直接检查的​​微生物游泳和趋化行为。 epifluorescence相衬显微镜结合使用允许的物理尺寸和营养补丁的扩散特性进行直接检查，同时观察在人口聚集的反应，除了个别微生物的游泳行为。这些实验显示，一些浮游植物，异养菌和phagotrophic原生生物物种，善于寻找和利用很短的时间框架内扩散微尺度资源补丁。我们还表明，中等剪切速率，海洋细菌能够争取自己的流量，并通过他们的环境中游泳。但是，超出阈值高剪切水平，细菌是对齐的剪切流动，并没有从流障碍游泳能力较差。微流体新颖，价格低廉的方法，为水生微生物生态学研究，并且由于其准确地建立切合实际的流场和基板梯度在微尺度的适用性，理想地适用于在互动的最小尺度微生物行为的考试。因此，我们建议微流体代表了获得一个更好地了解海洋中的微生物生态的有价值的工具。

Watch this Scientific Journal Video about Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers at JoVE.com

Meiosis  | Biology Video Collection

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

制造微流体通道和内修修补补的营养海景和剪切流内的细菌的游泳行为研究海洋微生物的趋化觅食的习性，他们在实验中实现。

海洋微生物的趋化反应的微观尺度的营养层

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

chemotactic-response-marine-micro-organisms-to-micro-scale-nutrient

Research

JoVE Journal

Biology

13.2K 次观看.  MIT - Massachusetts Institute of Technology. 制造微流体通道和内修修补补的营养海景和剪切流内的细菌的游泳行为研究海洋微生物的趋化觅食的习性，他们在实验中实现。

视频: Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

共生层 - 可视化的白蚁后肠微生物群落

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

科研

生物学

Micro-scale Engineering for Cell Biology

细胞生物学的微观尺度工程

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

在使用微深皮质层的活体成像

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

生存时间的测量<em&gt;臂</em&gt;轮虫：产妇条件同步

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水培：一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...