我们想感谢波特集团的成员，根据协议提供宝贵意见。这项工作是由临床研究培训奖学金从美国神经病学基金会学会CMH的，从国家普通医学科学研究所（NIGMS;奖学金<a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a> ）JDR（GM08169 ），授予来自美国国立卫生研究院国家神经疾病研究所和中风（NS054809），一个露丝属Kirschstein国家研究服务奖JDR（NS060392），EFRI授予美国国家科学基金会（0836017），库尔特基金会翻译研究奖，和一个翻译研究奖学金JDR（NS007480）。

<ol>
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CMH的有没有透露。<br /&gt; JDR和SMP支付公理生物系统公司的顾问。<br /&gt;这篇文章和视频提交的材料费和视频处理费慷慨赞助多通道系统。

此协议表明如何文化的指示，并维持神经网络的微电极阵列，以及录制和刺激神经网络的介绍。一些比较常见的问题，从多边环境协定的录像时，还讨论了在多边环境协定的故障排除部分。在协议中有偶尔的变化作为整个讨论和这些变化常常利用优化一定的实验设计所需的特定条件。 虽然这里介绍的是记录和刺激系统组成，设计与MCS前置NeuroRighter 15我们的自定义，有多个其他系统的神经网络，可以提供一个接口。选择一个特定的设置，将取决于具体的实验需要。 在这个协议提供了简单的记录和刺激的例子，然而，这些多边环境协定的文化，可用于提供有关突触活动和神经元网络连接复杂的问题的洞察力。如上所述，正在进行的工作是利用这种技术，像细胞的可塑性研究的过程，从我们的实验室最近出版的发展，兴奋性毒性和神经退行性疾病，例如6-10，表明重现性目标导向学习与实时闭环刺激在体外应对当前的神经网络活动3。 虽然多边环境协定的文化往往是挑剔的和微妙的，由于其简单性和可访问性（比较完整的动物），他们提供了一个强大的模型研究在网络层面的神经元活动。

用品<ul><li>一次性瓶顶过滤器（150毫升，0.2微米孔径; NALGENE＃565-0020） </li><li> 18超时怀孕的雌性大鼠胚胎一天</li><li>微电极阵列（在协议描述，B1） </li><li>微电极阵列盖（见描述协议，B3） </li><li>尼龙细胞过滤器（40微米，BD猎鹰，＃352340） </li><li>聚丙烯锥形瓶（无菌15毫升） </li><li>聚苯乙烯培养皿（无菌，35mm和100x15mm） </li><li>吸取提示（无菌，20μL，200μL和1000μl） </li><li>血清吸管（5毫升） </li></ul>设备： <ul><li>生物安全柜/层流罩</li><li>细胞计数</li><li>离心分离</li><li>冰的容器</li><li>解剖剪刀和镊子（消毒） </li><li>层流罩在解剖显微镜</li><li>手持式电动pipetman </li><li> Hemacytometer </li><li> Isofluorane和毒气室</li><li>光镜下</li><li>手持pipetman（P - 20（20μL），P - 200（200μL），P - 1000（1000μl）） </li><li> MEA录音系统（见协议时，D1的描述） </li><li>鼠类断头台</li><li>三气培养箱（见协议，B15的描述） </li><li>在引擎盖与吸引器连接的保温瓶</li><li>涡旋</li><li>水浴（35-37 ° C） </li></ul>解决方案和试剂： BSA溶液： 5％的牛血清白蛋白（SIGMA A - 9418）的磷酸盐缓冲液pH 7.4和0.2微米过滤。该解决方案可提前准备的时间和储存在4 ° C几个月。 细胞培养基： 结合90毫升贝科的修改鹰的介质（尔湾科学9024），10毫升马血清（Gibco公司16050），1毫升钠丙酮酸（100MM，Gibco公司11360），250μLGlutaMAX（Gibco公司35050），和胰岛素（终浓度为2.5微克/毫升，西格玛的I - 5500），然后通过一个0.2微米filter.22暖平衡在孵化器的细胞培养基，使用前。 夹层的解决方案，准备木瓜蛋白酶溶液： 23 <table border="1"><tr><td>夹层解决方案（100毫升股票） </td><td>结合起来，才能过滤0.22微米和存储，在4 ° C。 </td><td></td></tr><tr><td>组件</td><td>容ml </td><td>终浓度（毫米） </td></tr><tr><td>双蒸水</td><td> 91.175 </td><td></td></tr><tr><td> 1个M氯化镁2 </td><td> 0.58 </td><td> 5.8毫米</td></tr><tr><td> 1个M氯化钙2 </td><td> 0.025 </td><td> 0.25毫米</td></tr><tr><td> 0.5 M羟乙基</td><td> 0.32 </td><td> 1.6毫米</td></tr><tr><td> 0.5％酚红</td><td> 0.2 </td><td> （0.001％） </td></tr><tr><td> 0.1 N的氢氧化钠</td><td> 0.2 </td><td> 0.2毫米</td></tr><tr><td>中号硫酸钠</td><td> 4.5 </td><td> 90毫米</td></tr><tr><td> 1中号硫酸钾</td><td> 3.0 </td><td> 30毫米</td></tr><tr><td> 0.1 M的Kynurenic酸</td><td> 1.0 </td><td> 1毫米</td></tr><tr><td> 5 mM的APV </td><td> 1.0 </td><td> 0.05毫米</td></tr></table>该解决方案可提前准备的时间和储存在4 ° C几个月。 DNA酶我： Invitrogen公司（＃18047019）。 50μL等分闪光冷冻和储存在-80 ° C层流罩是一个等份解冻之前，要启动细胞的解离过程。 汉克的平衡盐溶液： 西格玛（＃4891）。一个1X解决方案是准备在去离子水，在0.2微米的无菌过滤，并保存在4 ° C。 Hibernate的解决方案： 培养每天做准备。再加上98毫升的L - 15培养基（Invitrogen公司11415）2毫升的B - 27的补充（50X，Invitrogen公司＃17504），在0.2微米过滤。 层粘连蛋白的解决方案： 培养每天做准备。稀释980μL细胞液浓度为0.02mg/ml最终20μL层粘连蛋白（1mg/ml的L - 2020，Sigma公司）。层粘连蛋白（1mg/ml的）20μL分装储存于-80 ° C和解冻之前使用。解冻应该出现在替补席上的顶部，而不是在水浴快速解冻，有时会导致层粘连蛋白的胶凝。 木瓜蛋白酶溶液：23 暖的10毫升（以上）的夹层解决方案，以30-32 ° C。新增200个单位的木瓜蛋白酶（Sigma公司P - 4762）和1.6毫克L -半胱氨酸（Sigma公司的C - 7880）。约14μL达到0.1 N NaOH溶液，调节pH值至7.4。静置约30分钟，直到溶液CL耳朵，过滤0.2微米，闪光灯在2毫升分装在15毫升无菌聚丙烯锥形瓶和存储液氮冻结在-80 ° C。在35-37 ° C开始之前协议解冻等份。 聚乙烯亚胺（PEI）的解决方案：13 爱德华王子岛的100μL（50％W / V，Sigma公司的P - 3143）是100毫升四硼酸钠缓冲液（0.1 M的pH值8.4，适马的B - 9876）稀释，终浓度为0.05％W / v的在0.2微米的无菌过滤。这可以提前做好准备，并在4 ° C储存几个月。 无菌去离子水： 高压灭菌器和允许冷却至室温，在使用前。 胰蛋白酶（0.25％EDTA，GIBCO 25200）： 1毫升等分闪光冷冻和储存在-80 ° C等分在35-37℃的水浴使用前解冻。

how to culture, record and stimulate neuronal networks on micro-electrode arrays (meas)

对于上个世纪，世界各地的许多神经学家都献出生命的理解神经网络是如何工作的，为什么他们停止在各种疾病的工作。研究包括神经病​​理学观察，荧光显微镜与遗传标记，并在分离的神经元和切片准备细胞内记录。该协议将讨论另一种技术，其中包括日益增长的微电极阵列（也称为多电极阵列，多边环境协定）的游离神经文化。 使用这个系统比其他技术有多种优势。多边环境协定的游离神经文化提供了一个简化的模型中，网络活动可以通过阵列的多个电极电刺激序列操纵。因为网络是小的，刺激的影响是有限的，以观察到的领域，这是不完整的准备工作的情况下。在决策在形态上容易的变化监测各种成像技术单层细胞的生长。最后，文化上的多边环境协定可以存活超过一年的体外删除任何明确的时间限制，与其他养殖技术固有1 。 我们的实验室和世界各地的人都在利用这一技术，询问有关神经网络的重要问题。此协议的目的是提供必要的信息，为设立，关心，录制和电刺激对多边环境协定的文化。体外网络提供生理有关的问题，要求在网络和细胞水平，从而更好地理解的一种手段大脑功能和功能障碍。

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How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

该协议提供了必要的成立，关心，录制和电刺激对多边环境协定的文化信息。<em在体外</em&gt;网络提供了一个生理有关的问题，要求在网络和一个更好地理解大脑功能和功能障碍导致的细胞水平的手段。

如何以文化，记录和刺激神经网络的微电极阵列（多边环境协定）

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

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How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

54.3K 次观看.  Emory University School of Medicine. 该协议提供了必要的成立，关心，录制和电刺激对多边环境协定的文化信息。

视频: How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

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 ...

科研

生物学

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

小鸡前OVO文化前OVO CAM含量：它是如何真正起作用

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

复用的恒化器阵列的设计与应用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

研究蛋白质和微RNA调制对脂肪细胞功能的影响的脂肪细胞培养模型

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...