Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1，准备工作<ol><li>将实时细胞分析仪的台在组织培养箱设定为37℃，并用5％的CO 2。完成所有的细胞培养操作和药物治疗在无菌条件下，组织培养罩。 </li><li>注:神经元细胞系中，需要不同的温度设置进行比较，以对培养的标准条件下，应作相应的调整。对于弱粘附细胞系，用包衣剂，以促进细胞的金微电极中的电子板96的孔的底部的相互作用。 </li><li>制备1,000倍原液的药理化合物用于细胞培养的治疗（神经保护剂或第二信使通路的抑制剂，在适当的溶剂 - 二甲亚砜或无菌水），并将其存储在-20℃。使用溶剂作为车辆在以下的处理。 </li><li>培养人成神经细胞瘤SK-N-SH细胞在DMEM / F12培养基中在细胞培养烧瓶中的10％胎牛血清（FBS）（增殖培养基）175 cm 2的表面到约75％的密度。保持一个范围（约10次传代）中的细胞传代次数，以确定在细胞增殖和响应于细胞毒性率方面的实验之间的一致性。最好是通过较低的数字。 </li><li>用PBS洗涤附着的SK-N-SH细胞层，并trypsinize与在37℃5分钟，0.05％胰蛋白酶-EDTA溶液。离心将细胞在170×g下5分钟以除去胰蛋白酶。悬浮细胞在10毫升增殖培养基。计数细胞与权杖细胞计数，并通过与增殖培养基稀释细胞调节细胞数至300,000个细胞/ ml。 </li></ol> 2，电镀和SK-N-SH细胞增殖<ol><li>加入100μl扩散介质向E-Plate 96上的每个孔中，将其放置在组织培养罩在室温下平衡30分钟。将E-解放军TE 96中在37℃下的实时细胞分析站中的CO 2培养箱中培养。 </li><li>启动实时细胞分析软件。在布局页面选择包括在实验孔中，并在编辑框中输入有关的细胞类型，细胞数目，名称和用于细胞治疗的化学化合物的浓度的信息。 注:在本协议的目的，至少4个重复，建议每次治疗。 </li><li>在计划软件页确定包括在该实验中，通过选择扫描测量细胞指数和扫描的每个步骤之间的时间间隔的数目"步骤"。自第1步是预定为背景测量，选择"添加步骤"，并设置第2步以测量细胞指数每隔15分钟进行96小时。 注:对于治疗，其中具有快速反应动力学的影响，预计在较短的时间间隔设置扫描。 </li><li>单击开始启动自动预定第1步和测量介质的背景阻抗。此值由软件自动在每个数据点减去一旦细胞被加入到孔中。 </li><li>使用300,000个细胞/ ml的先前在步骤1.5的方法制备）的细胞悬浮液中的扩散介质。 30,000个细胞/孔的细胞毒性/神经保护研究和15,000个细胞/孔细胞增殖的研究。取出电子盘，加入100微升细胞悬液，每孔的细胞毒性/神经保护作用的研究，加入50微升细胞悬液和50微升增殖培养基，每孔细胞增殖的研究。需要细胞每孔接种的数量必须为每个细胞系凭经验优化。 </li><li>轻轻摇动电子盘96连电镀细胞。离开E-板96在组织培养罩在RT以使细胞沉淀均匀地向孔的底部30分钟。 </li><li>插入电子板96中的实时细胞分析站，并开始在计划页的步骤2年龄。通过查看绘图页面和软件的细胞指数页面上的原始细胞指数数据在细胞曲线监测整个实验细胞指数值。 </li></ol> 3，血清剥夺<ol><li>电镀细胞24小时后，暂停该实验中，并从实时细胞分析站取出电子板96。稀从1,000X股第二信使抑制剂的DMEM / F12无血清介质 - 以200×的最终浓度。治疗细胞的第二信使通路1μl的抑制剂，以用于其在神经/神经保护前30分钟开始的细胞毒作用的参与进行测试，以抑制各通路。返回电子盘96台，恢复实验细胞指数的测量。 </li><li> 30分钟后，再次暂停实验。小心地从电子盘全面清除增殖培养基中96井用移液管（或者多道移液器），注意不要破坏贴壁细胞层通过将移液管尖的边缘到阱的角落。加入200μl/无血清的DMEM / F12培养基（血清饥饿培养基）中的井。确保细胞培养基的快速交换，以最小化细胞的机械搅拌。 </li><li>稀释的化合物其从1,000X股票神经保护作用的无血清DMEM / F12培养基，以200×的最终浓度进行试验。加入1μl化合物对于其治疗作用和对第二信使通路中的各个孔中加入1μl酶抑制剂根据实验方案进行测试。 </li><li>在细胞增殖的研究并没有改变中。稀释的化合物可以从1,000X库存进行测试，以200×在增殖培养基中的最终浓度，治疗与每孔1微升，并继续培养中增殖培养基。 </li><li>恢复实验继续进行细胞指数的测量，每15分钟进行96小时为预先设定的。 </li></ol> 4，数据Analysis <ol><li>对于神经毒性/神经保护数据的标准化细胞指数，以药物治疗或换液前的最后一个时间点，选择"正常化细胞指数"，以减少实验之间的差异和"规范化时代"的情节页面上。 </li><li>突出显示绘图页上的井感兴趣的实验条件，然后点击"添加"来绘制归一化细胞指数曲线。观察的神经毒性/神经保护作用的，或第二信使抑制作为归一化的细胞指数曲线作为时间的函数的动力学。 </li><li>观察细胞指数曲线测试的药物治疗在增殖培养基中作为时间的函数来评估是否参与了神经保护/神经毒性作用，对细胞增殖的效果。 </li><li>出口从细胞指数的软件页面中的所有细胞指数的时间点实验信息到一个Excel文件来启动结果进行统计评估<br/&gt;注意:由于在统计分析的第一步了一组用韦尔奇的t检验，以P值修改MATLAB程序绘制semilogarithmically，利用对时间尺度倒轴（作为辅助代码文件）。这些程序允许检测的p值在时间过程的整个实时细胞分析实验，从而帮助的时间点更详细的统计分析的选择。请参阅有关的程序的详细信息的补充材料。 </li><li>对于不同的处理条件下在特定时间点在细胞指数值的差异的统计分析，在从该导出的Excel文件中各个时间点选择细胞指数值。具有单向或双向方差分析（ANOVA），接着利用统计软件的事后检验分析所选择的时间点的细胞指数值。 </li></ol>

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当前视频文章出版费用由"ACEA Biosciences公司"进行了赞助。

当前协议提出了一种实时的细胞分析仪的实用程序，在连续和无标记的条件下评估的化合物在神经元细胞系中的神经保护/神经毒性作用，并深入了解所涉及的效果的第二信使途径。 即使在实时细胞分析仪的实用程序来研究细胞毒性和药物对细胞增殖的影响一般认为，只有很少的研究已经在神经元相关的细胞类型中使用它。我们先前的研究显示了实时细胞分析仪的实用程序来?...

神经细胞死亡起到许多脑相关疾病1的病理生理学中起关键作用。的可靠和高通量的体外毒性试验的可用性是至关重要的，以获得更好的洞察神经毒性的作用机制并帮助选择神经保护分子作为治疗候选药物开发2。不过，也有不少限制使用最广泛的体外神经毒性assays.They评估神经毒性/神经保护在单一时间点不让动力学拆分;经常使用的标签或探针可与信号通路干扰，并在同一细胞群限制进一步的研究，并且通常是劳动密集型的，并且在许多情况下，不提供机械性的认识。在本研究中我们展示了一个实时的阻抗为基础的细胞分析仪的实用程序来确定神经毒性和神经保护作用的实时，并在神经元细胞系无标签条件，并通过所涉及到的作用的第二信使途径的分析洞察下游机制。 先前的研究已经证实了实时细胞分析的有效性，以确定在标准技术3,4,5,6对比的细胞毒性以及对细胞系的细胞增殖的影响。例如，在标准细胞存活力WST-1法的读数与细胞指数值之间观察到几个时间点下基底扩散条件和HeLa细胞3两种不同的有毒范式后的良好相关性。在A549和MDA-MB-231细胞增殖和细胞毒性挑起与微管 ​​稳定剂紫杉醇表现出非常相似的值时，通过细胞指数测量，评估和标准地使用磺酰罗丹明B（SRB）法4。在永生的海马神经元的神经细胞系HT-22细胞指数测量，验证了自己的能力吨ö检测细胞增殖，谷氨酸毒性和细胞保护作用对广泛使用的3（4,5 -二甲基-2-基） - 2,5 -二苯基溴（MTT）法5。在同一研究中的MTT法检测结果和细胞指数测量也由泛Caspase抑制剂QVD 5个测量神经元祖细胞的增殖，细胞毒性后的生长因子剥夺和细胞毒性的救援很好的相关性。细胞毒性通过凡德他尼（血管内皮生长因子受体和表皮生长因子受体抑制剂）诱导的NIH 3T3细胞显示出与细胞指数值或中性红摄取试验6测定结果相似。 我们最近使用的实时细胞分析系统，以评估血清素2A的神经保护作用（5-HT 2A）受体激动剂（±）-2,5 -二甲氧基-4-iodoamphetamine盐酸盐（DOI）的神经元细胞系（ SK-N-SH细胞），并筛选山高的参与通过监测上观察到的神经保护7的化学抑制的效果第二信使通路。有趣的是，对5-HT 2A受体具有两个致幻和nonhallucinogenic激动剂（如DOI和麦角乙脲，分别），其可以激活两个共同和不同的第二信使通路8。 所提出的技术的优点是，它允许收集在天的过程中对细胞存活的实时信息，划定第二信使途径参与其中，以评估增殖效应的神经保护作用的可能贡献，并选择一个最佳的时间为在相同的细胞群附加端点研究。在当前的协议的工作流程的示意图示于图1。

<table><tbody><tr><td>xCELLigence&amp;nbsp; RTCA SP system bundle</td><td>ACEA Biosciences</td><td>No: 00380601030</td><td>Consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td></tr><tr><td>E-plate 96</td><td>ACEA Biosciences</td><td>No: 05232368001</td><td>For culturing the cells, inserted in the RTCA SP Station</td></tr><tr><td>SK-N-SH cells</td><td>ATCC (in partnership with LGC Standards)</td><td>HTB-11</td><td>Can be replaced by another adherent neuronal cell line of interest</td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8437</td><td>Cell culture medium</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>16140-063</td><td>Supplements proliferation, but not serum deprivation medium</td></tr><tr><td>Tissue culture flask T175</td><td>Sarstedt</td><td>83.1812.302</td><td>For culturing cells, which will be later plated on the E-Plate 96</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td>For trypsinization of cells cultured in tissue culture flasks</td></tr><tr><td>Scepter 2.0 cell counter</td><td>Merck Millipore</td><td>PHCC20060</td><td>Automated cell counter</td></tr><tr><td>Phosphate-buffered saline</td><td>Life Technologies</td><td>10010-015</td><td>For washing the cells</td></tr><tr><td>(&amp;plusmn;)-DOI hydrochloride</td><td>Sigma-Aldrich</td><td>D101</td><td>5-HT2A agonist for cell culture treatment</td></tr><tr><td>LY-294002 hydrochloride</td><td>Sigma-Aldrich</td><td>L9908</td><td>PI3-K inhibitor for cell culture treatment&amp;nbsp;</td></tr><tr><td>Lisuride maleate</td><td>Tocris Bioscience</td><td>4052</td><td>Compound with 5-HT2A agonistic activity for cell culture treatment</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D4540</td><td>For dissolving LY-294002 and lisuride maleate</td></tr></tbody></table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

许多大脑相关的疾病具有参与其病理生理学的神经元细胞死亡。改进的体外模型来研究药物和涉及将有助于深入了解神经保护/神经毒性的分子机制和可能潜在地促进药物开发下游途径的神经保护或神经毒性作用。然而，许多现有的体外毒性实验有很大的局限性-大多数评估神经毒性和神经保护在一个单一的时间点，而不是让观察的时间过程和效果动力学。此外，机会来收集关于涉及神经保护中的实时下游信号传导途径的信息是非常重要的。在当前的协议中，我们描述了使用实时的基于阻抗的细胞分析装置，以确定血清素2A（5-HT 2A）受体激动剂在神经元细胞系在无标记和实时康迪特治疗作用使用阻抗测量离子。此外，我们证明，第二信使途径的抑制剂可用于描绘参与神经保护作用的下游分子。我们还描述了该技术的实用程序来确定对细胞增殖的效果，是否有助于所观察到的保护作用。该系统采用称为E-平板的包含交替的孔的底部表面的金微电极阵列上，作为细胞传感器专用微电子板。阻抗读出是通过贴壁细胞，细胞生存力，形态和粘附的数量进行修改。一种称为细胞指数无量纲参数是从所述电阻抗测量得到和用于表示该细胞的状态。总体而言，实时基于阻抗的细胞分析装置允许进行实时，神经保护和神经毒性的无标记的评估，以及第二信使途径参与了评价，有助于更在体外的潜在的神经保护化合物的详细的和高通量的评估，选择治疗的候选人。

<html> 血清剥夺导致在细胞指数值，它可以连续地实时细胞分析仪进行监测，以减少 神经刺激的细胞导致的降低细胞指数值，它可在实时的呈现技术和动态这是依赖于特定的神经刺激和研究的细胞类型进行监测。 图2显示了增加的当SK-N-SH细胞生长在增殖培养基中，直到达到平台（绿线）和细胞指数的下降之后稳定在新的水平较低的开关的细胞血清剥夺...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

real-time-impedance-based-cell-analyzer-as-tool-to-delineate

Research

JoVE Journal

Neuroscience

11.4K 次观看.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

视频: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

准备和测试阻抗为基础与RTgill-W1细胞饮用水进行快速评估水样进行毒性射流生物芯片

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

科研

环境科学

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

用于实时细胞分析仪的阵列金电极再生

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

生物化学

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

发育生物学

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 ...

生物学

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

神经科学

通过电电池-衬底传感评估 MOF 诱导的电池行为变化

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

作者聚焦:评估人肺上皮细胞对金属有机框架反应的进展 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

用于实时评估金属有机框架毒理学特征的电池-基底传感

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

生物工程

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

摘要

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