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1。猫星形胶质细胞的细胞培养和治疗与H 2 O 2 <ol><li>文化细胞系G355 - 5在75厘米的 2瓶用DMEM媒体加营养素在37 ° C和5％的CO 2，直到60％融合（〜5天）。每1-2天更换介质或需要保持一个健康的文化。 </li><li>取出介质，加2毫升0.25％胰蛋白酶。电梯45秒吹打细胞 - 在室温（RT）1分钟。 </li><li>快速传输悬挂一个15毫升的锥形管中，含有10毫升的媒体。轻轻混匀。 </li><li>离心3分钟，23℃在300 XG获得沉淀。上清液丢弃，而不会干扰颗粒。 </li><li>添加6-7毫升的新鲜媒体和悬浮细胞。 </li><li> 〜1毫升的细胞，每孔6孔板。加入2-3毫升媒体，每口井和孵化，直到80-90％融合。 </li><li>准备在媒体100μm的过氧化氢 ​​溶液（H 2 O 2）。 </li><li>从每口井的媒体和替换要么是2毫升的新鲜传媒（对照组），2毫升H 2 O 2（治疗）或DPBS 2毫升。孵育于37℃，5％的CO 2为3小时。 </li><li>删除的解决方案，并添加1毫升的胰蛋白酶解除细胞。细胞转移到1.7 ml的Eppendorf管中含有0.5毫升DPBS。这一步应该是每孔单独执行，以防止长时间的胰蛋白酶孵育细胞死亡。 </li><li>旋转300 XG为3分钟，弃上清，悬浮在1毫升的DPBS。 </li></ol> 2。染色活性氧<ol><li>以最小的光线照射，以防止漂白，染色的所有步骤应进行。 </li><li>加入1μL50毫米CCCP的阳性对照。在37℃，5％CO 2为5分钟。 </li><li>添加5μL10毫米CM - H 2 DCFDA解决的H 2 O 2组。在37℃，5％CO 2为15-30分钟。无染色添加到生理盐水样品。 </li><li> 2毫升的DPBS清洗样本，以清除残留的污点。 </li><li> 3分钟，在300 XG旋获得沉淀。 </li><li>重悬在0.5毫升的DPBS细胞。每个样品中加入1μL的PI，除盐水。 </li><li>样品转移到5毫升流式细胞仪管。 </li><li>流式细胞仪上运行。 </li></ol> 3。流式细胞仪<ol><li>运行样品上贝克曼FC500配备与488 nm的氩激光及以下带通行证（或同等学历）：525，775和620 nm，所有± 20纳米。 </li><li>准备适当的控制：未染色细胞，CM - H 2 DCFDA染色只，有价证券，双染色（阴性对照）。 </li><li>做一个协议具有以下histoplots：FS与SS，有价证券与CM - H 2 DCFDA。它也应该有以下直方图：细胞的数量与FL1，FL2，FL3，对应的污渍。 </li><li>在运行样品，检查设备，液体和废物容器。至少20分钟的热身设备。 </li><li>清洁设备，并执行适当的珠子，根据厂家的标准校准。 </li><li>选择您的协议，并运行这些示例。 </li><li>清洁设备，完成以前。 </li><li>清洁真空根据厂家的标准线。 </li><li>关闭流和清洁头/真空。 </li><li>关闭软件和计算机。 </li></ol> 4。代表性的成果： 流式细胞仪检测结果进行了分析使用FlowJo 7.6和色斑的控制客观上设置的门控。在此基础上，健康的细胞出现直方图和活性氧（氧化应激）左侧是检测一个细胞转移到右侧。图1显示了健康猫的星形胶质细胞的过氧化氢的影响的结果。从FL1的数据是用来衡量CM - H 2 DCFDA，这表明存在ROS的力度。正如预期的那样，一个金额较高的活性氧与H 2 O 2处理的样品检测。这是显示在荧光强度转向由左到右的直方图。比较健康细胞用DMEM与DPBS治疗时，并没有显着的变化ROS的量（图2）。 <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="图1"/> 图1流的健康猫的星形胶质细胞的氧化应激水平和对健康细胞的H 2 O 2的效果的流式细胞仪的结果。结果表明，H 2 O 2增加在健康细胞中的ROS水平相比，与没有治疗的细胞（DMEM） 。 <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="图2"/> 图2。流量孵育DPBS 3小时的健康细胞中检测到ROS水平的流式细胞仪结果。

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通常建议的病毒的机制诱导神经元的损伤是氧化应激1，4，7，9，13，15，16，18-22，27，31 。基本上，它是通过病毒暴露的神经胶质细胞（星形胶质细胞和小胶质细胞）释放，如氧化钾，超氧阴离子自由基，一氧化氮，和/或过氧化氢，这是有毒的神经细胞的活性氧自由基。同样，氧化应激也被作为一个主要的甲基苯丙胺神经毒性2，6，17的病理机制的建议。由于本集团在病毒和药物?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM培养液</td><td> Cellgro </td><td> 15 - 013 - CV </td><td></td></tr><tr><td>纪念维生素</td><td> Cellgro </td><td> 25 - 020 - CL </td><td> 1％（V / V） </td></tr><tr><td> L -谷氨酰胺</td><td> Hyclone公司</td><td> 17 - 605E </td><td> 1％（V / V） </td></tr><tr><td>非必需氨基酸</td><td> Hyclone公司</td><td> 25 - 025 - CL </td><td> 1％（V / V） </td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30071.03 </td><td> 20％（V / V） </td></tr><tr><td>人体必需的氨基酸</td><td> Cellgro </td><td> 25 - 030 - CL </td><td> 0.2％（V / V） </td></tr><tr><td> 500毫升真空过滤系统</td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml锥形管</td><td>鹘</td><td> 352097 </td><td></td></tr><tr><td> 75厘米2组织培养瓶</td><td>鹘</td><td> 353136 </td><td></td></tr><tr><td> 6孔细胞培养板</td><td>鹘</td><td> 353224 </td><td></td></tr><tr><td> CM - H 2 DCFDA </td><td> Invitrogen公司</td><td> C6827 </td><td></td></tr><tr><td>碘化丙啶（PI） </td><td>西格玛</td><td> P4170 - 10毫克</td><td></td></tr><tr><td>胰蛋白酶</td><td>龙沙</td><td> 17 - 160F </td><td></td></tr><tr><td> H 2 O 2 </td><td>西格玛</td><td> H6520 </td><td></td></tr><tr><td> HYQ抗生素</td><td> Hyclone公司</td><td> SV30079.01 </td><td> 0.1％（V / V） </td></tr><tr><td> G355 - 5细胞</td><td> ATCC </td><td> CRL - 2033 </td><td>正常猫科动物的大脑</td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

的病毒通常建议的机制，诱导神经元的损伤是氧化应激。星形胶质细胞有重要作用，在控制对中枢神经系统（CNS）的氧化应激。星形胶质细胞有助于保持神经元以及保护神经元的活性氧（ROS）的稳态环境。 CM - H2DCFDA是一个细胞的活性氧的存在透水的指标。 CM - H 2 DCFDA作为一个非荧光化合物进入细胞，并成为荧光细胞酯酶删除后的醋酸组，该化合物是氧化。细胞，流式细胞仪检测，发现绿色荧光细胞处于氧化状态的指示。 CM - H 2 DCFDA是由不同的ROS的大量氧化易感。这种缺乏特异性，对活性氧可以氧化CM - H 2 DCFDA，使该化合物为使用宝贵的摄政在一个发病调查的早期阶段，这种检测可以用于氧化的细胞环境到屏幕上，无论是其中的氧气自由基或活性氧相结合是负责细胞的条件。一旦它被建立，活性氧是目前CM - H 2 DCFDA氧化，然后可以进行更多的实验来确定在特定的发病过程中所涉及ROS或组合罗斯。这项研究的结果表明，与过氧化氢 ​​增加CM - H 2 DCFDA的加入荧光检测与生理盐水对照，表明这个实验是一个宝贵的测试，检测G355 - 5细胞内的氧化环境猫星形胶质细胞株。

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

一种检查方法检测细胞氧化的环境，来衡量的CM - H2DCFDA氧化。一旦氧化细胞内，CM - H2DCFDA的变化，从非荧光一​​种荧光的化合物。是用流式细胞仪检测荧光的这种变化，并表示在氧化环境中的细胞数量。

筛选法测定氧化应激在猫的星形胶质细胞的细胞线，G355 - 5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

screening-assay-for-oxidative-stress-feline-astrocyte-cell-line-g355

Research

JoVE Journal

Neuroscience

14.3K 次观看.  Western University of Health Sciences. 一种检查方法检测细胞氧化的环境，来衡量的CM - H2DCFDA氧化。一旦氧化细胞内，CM - H2DCFDA的变化，从非荧光一​​种荧光的化合物。是用流式细胞仪检测荧光的这种变化，并表示在氧化环境中的细胞数量。

视频: Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

科研

神经科学

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

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

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

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 ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...