笔者要感谢保罗·维悌希博士和吉纳维芙方女士的mRNA的提取和PCR援助，以及加内特凋谢和罗德尼·威廉斯纪念基金会的支持。

伦理学声明：下面列出的所有程序批准了悉尼动物伦理委员会的大学。 1.动物注：1和8 - 9个月大的小鼠（C57 / BL6）是从动物资源中心（珀斯，澳大利亚）中获得。老鼠被安置在博世灭鼠设施，在悉尼大学。 <ol><li>家鼠在上一个12/12小时亮/暗循环以获得食物和水随意标准鼠标笼中。 </li><li>鸿沟小鼠各年龄组为近红外（NIR）治疗，或假处理（对照）组进行比较。 </li></ol> 2.近红外（NIR）照射及深水治疗<ol><li>剃鼠标的头部和颈部区域的毛皮用电动剃刀尽可能防止头发快速再生完成治疗方案的前。为了保持小鼠的无病原体状态，使用70％的乙醇剃须动物，并从不同的笼子里的动物之间的F10兽用消毒剂清洗之间的乐器。这样做2 - 3天前开始治疗等动物不是过度处理。 </li><li>抑制小鼠握住尾部的近端，并允许它在用一只手或台式手掌放松，以便尽量减少应力，这可能会导致错误的结果。 </li><li>持有NIR（670 nm）的LED（发光二极管）器件1 - 2厘米的距离暴露（光头）区和开关设备上的90秒（ 图1A）。 注意：温度变化的90秒曝光结果测定为&lt;0.2℃下在100毫升的水中。 </li><li>对于假治疗组动物的2.3，但离开该装置关闭（ 图1B） -重复步骤2.2。 </li><li>重复步骤2.2 - 2.3对NIR阻断治疗组的动物，但覆盖器件有铝箔。 </li><li>每天approximat 2.5 - 重复步骤2.2E 24小时的间隔，连续5天。 </li><li>提取两个耳朵上的第五天后处理前庭感觉上皮（3嵴和1个椭圆囊斑）（见下文第3节 ）。 </li></ol> 3.组织提取20 <ol><li>制备300毫升基于甘油的人工脑脊液（ACSF）的组成（以mM计）：26的NaHCO 3，11葡萄糖，250甘油，2.5氯化钾，1.2的NaH 2 PO 4，1.2的MgCl 2，和2.5 氯化钙 。在加入氯化钙 ，燃气与卡波金（95％O 2和5％CO 2）的溶液来建立的pH为7.4，并避免钙沉淀（混浊）。寒意在-80℃冷冻该溶液45分钟，以使冰浆料形成。 </li><li>准备在标螺杆顶端微管的RNA分离裂解缓冲液（停止盖子的爆裂时冻结和解冻在液氮之间管压力的变化），根据制造商或通常的做法实验室的指示。有液氮准备在铝杜瓦瓶中。 注：液氮操作时，使用的防护服和eyeware。确保使用液氮在一个通风良好的房间作为其气体形式的体积大约是700倍，它的液体形式，并可能导致窒息。 </li><li>通过腹膜内注射深深地麻醉小鼠用氯胺酮（400毫克/千克）。允许后肢反射完全消退作为指示小鼠完全麻醉。 </li><li>杀头的小鼠用锋利的不锈钢剪刀，并沿着用刀片头骨的矢状皮肤切口（四舍五入＃22）。在这一点上和整个步骤3.5 - 3.9，保持颅，脑和潜在的前庭器官尽可能凉爽通过在组织常规应用冰冷学联的。 </li><li>使用标准模式剪刀尖手臂做出SMaL公司升切口头骨的Lambda和沿矢状缝切割。 </li><li>轻轻滑动的浅表骨钳一个臂顶骨保持刀片尽可能接近到骨头的下表面，而不拖动大脑下方。安全和扯远了顶骨横向和枕骨后部，直到大脑暴露。 </li><li>用一个不锈钢小铲子和解除大脑远离前部和中颅窝暴露前庭耳蜗神经（CN八）。横切神经以防止在该初级传入轴突直接支配前庭毛细胞不必要的紧张。 </li><li> CN八横断后取出大脑全盘 。 </li><li>观察含耳蜗和中颅窝周围前庭器官的骨迷路。让两个小切口，每个骨迷路旁边，握住前半规管和后拉切除整个结构盟友。 </li><li>立即浸入切除迷宫中含有冰冷ACSF溶液（如在步骤3.1中所述），同时连续地卡波金灌注解剖盘中。 </li><li>在立体显微镜下，按住迷宫由耳蜗并将其固定钳盘的底部。 <ol><li>直接使用细镊子划伤小开在上面的前半规管（SSC）壶腹骨。 </li><li>轻轻达到立即骨下方，向外弹离壶腹放大此开幕。谨慎行事这里没有推入钳开放和破坏脆弱的膜迷路下方。继续以这种方式，直到椭圆囊，前部和侧壶腹都暴露出来。如果可能的话删除后壶腹也。 </li></ol></li><li>用细镊子，轻轻抬起囊，并从骨迷路壶腹距离，直到他们完全不沾边。如果可能的话，他们持有由它们相关联的半规管，以避免损坏感觉上皮。在一些情况下，半圆形膜质导管的近端部分可能需要与虹膜剪切以从骨释放壶腹。 </li><li>牢固地抓住镊子的顶端和地点之间的前庭器官到先前在步骤3.2中制备的裂解缓冲液。轻轻绕摇动镊子在缓冲，以确保前庭器官已经从钳子脱离。检查，这是通过使钳子的立体显微镜下的情况。 <ol><li>拧上微管盖，并立即冷冻在液氮中的样品。 </li></ol></li></ol> 4. RNA提取和RT-PCR <ol><li>根据制造商的说明或优选的实验方案遵循信使RNA（mRNA）的提取的标准方法。 注：利用载体分子RNA，以帮助隔离mRNA的小产量商品化试剂盒在此使用协议。低洗脱体积可用于增加最终浓度的mRNA。 注：负“无酶”的控制（NEC的）和“无DNA模板”对照（NTC却）也必须被完成，以确保观察到的效果的有效性。 </li><li>适用的mRNA逆转录，以互补的DNA的靶基因的21-23（cDNA中）和扩增的标准方法。 </li></ol>

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作者宣称，他们没有竞争的财务权益。

这里所描述的代表性的结果表明，当与假治疗的小鼠相比较简短颅递送NIR光的（90秒/天5天）足以提高抗氧化剂的表达在老年小鼠的水平。而发射的热可以代表线粒体和/或神经元激活的一个来源，据报道为大鼠前庭传入神经​​24 -我们的热量由NIR发射测量LED器件是&lt;0.2℃，90秒，因此不太可能导致这里描述的变化。此外，与报告所强调以上，这里使用的NIR治疗不直接向感觉上皮或前庭传?...

余额递减性能和后续的瀑布是常见的，而自然老化1的不幸往往定义功能。这种下降的影响，既可以是自然和社会，和显著降低生活质量的老年人。作为回应，物理治疗和康复一直是研究的重点为瀑布，但尚未有一致的减少重复跌倒的发生率有关。与此同时，工作调查在周边或中央前庭系统（负责保持平衡的系统）是稀少，并且针对这些系统的潜在的治疗策略的变化和不平衡有限的根本原因。 在年龄相关的神经变性病症，包括年龄相关性黄斑变性2-4，阿耳茨海默氏病模型5-8，和帕金森氏病9-12最近的工作已经表明SI的神经保护作用mple非侵入性的应用的近红外（NIR）光。另外，在前庭系统，NIR已被用于提高在体外 13前庭初级传入神经 ​​元的活性。而没有很好理解NIR光的机构，利用NIR大多数的研究表明，NIR刺激线粒体复合物IV（细胞色素C氧化酶）14-17，以促进细胞代谢。在前庭感觉上皮I型毛细胞的表皮下板是致密的线粒体18，因此可以代表用于治疗NIR治疗作用的部位。 这里，经颅的简要的，非侵入性的治疗方案应用于NIR可用于测量细胞代谢（和暗示线粒体功能）在小鼠前庭感觉上皮进行说明。还讨论了为制备的前庭感觉上皮和它表明NIR增加一个ubiquito的表达我们的抗氧化剂（超氧化物歧化酶1）中的感觉上皮-先前证明是对耳蜗毛细胞存活19重要。

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			<td height="63" style="height:63px;width:216px;">Ketamine</td>
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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

策略随着年龄的增加衰减下降平衡功能主要是专注于物理疗法，包括平衡工作和锻炼。然而，这些方法没有解决平衡下降的根本原因。使用小鼠，近红外光（NIR）的细胞上，在前庭感觉上皮的代谢的影响进行了评估。收集的数据表明，这种简单和安全的干预可能保护这些脆弱的细胞从自然老化的有害影响。的mRNA从分离的前庭外周感觉上皮（壶腹嵴和椭圆囊斑）萃取，随后转录成cDNA文库。这个库然后探查无处不抗氧化剂（SOD-1）的表达。抗氧化剂的基因表达，然后用于量化细胞代谢。用年轻的经颅交付NIR（4周）及以上（8 - 9个月）小鼠，并简要治疗方案（90秒/天5天AYS），这项工作表明NIR单独可足以改善线粒体功能的前庭感觉上皮。由于目前没有可用的，负担得起的，非侵入性的治疗方法，提高前庭毛细胞功能，外部NIR辐射的应用程序提供，以抵消对细胞在矿井前庭感觉上皮代谢老化的影响，潜在的战略。

比较近红外治疗的年轻年长的影响（4周）和（8 - 9个月）小鼠，我们测得的抗氧化剂超氧化物歧化酶1（SOD-1）的表达年轻的（N = 16）和老年人（N = 20）小鼠是NIR治疗，假治疗，或NIR封; 图2示出了相比于年轻假处理的动物一个显著增加β肌动蛋白标准化的SOD-1的表达超过2倍于年轻NIR处理的动物（P &lt;0.01），年轻的NIR阻断动物（P &lt;0.01）。较旧的NIR处理的动物也显示出超过一个时与旧NI...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

线粒体功能障碍是细胞衰老的标志。本文使用的非侵入性的近红外（NIR）处理，以改善在老化小鼠前庭感觉上皮线粒体功能。

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.8K 次观看.  University of Sydney. 线粒体功能障碍是细胞衰老的标志。本文使用的非侵入性的近红外（NIR）处理，以改善在老化小鼠前庭感觉上皮线粒体功能。 

视频: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

近红外光谱技术在婴儿，儿童及成人的认知功能探索

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

科研

生物学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...