著者は、mRNA抽出とPCR、およびサポートのためのガーネットパッシーとロドニー·ウィリアムズ記念財団との援助のためにポール博士分かっていると氏ジュヌヴィエーヴ·フォンを認識したい。

倫理声明：以下のとおり、すべての手順は、シドニーの動物倫理委員会の大学によって承認された。 1.動物注：1と8から9カ月齢のマウス（C57 / BL6）は、動物資源センター（パース、オーストラリア）から入手した。マウスは、シドニー大学のボッシュ齧歯類の施設に収容した。 <ol><li>食料と水を自由にアクセスできる12/12時間の明/暗サイクル上の標準的なマウスケージの住宅マウス。 </li><li>近赤外線に各年齢群で除算マウス（NIR）処理された、または偽の比較のために（対照）処置群。 </li></ol> 2.近赤外（NIR）照射とシャム治療<ol><li>治療レジメンの完了前に髪の迅速な再成長を防ぐために可能な限り厳密に電気かみそりでマウスの頭頸部領域に毛皮を剃る。マウスの病原体を含まない状態を維持するために、70％エタノールを使用してシェービング動物や別々のケージから動物との間にF10獣医消毒剤の間に楽器をきれいにする。動物が過剰に処理されないので、治療前の先頭に3日間 - この2か。 </li><li>尾の近位端を保持することによってマウスを拘束し、誤った結果をもたらし得る応力を最小限にするように、一方またはベンチトップの手のひらにリラックスできるようにする。 </li><li> LED NIrを（670 nm）を（発光ダイオード）1デバイスホールド- 2センチ離し暴露（坊主頭）地域からの90秒（ 図1A）のための装置のスイッチを入れる。 注：90秒の曝露の結果として温度変化を100mlの水に0.2°C &lt;として測定された。 </li><li>動物の偽処置群について2.3が、デバイスがオフになった（ 図1B）を残す-繰り返して、2.2を繰り返します。 </li><li>動物の近赤外ブロックされて治療群2.3が、アルミ箔でデバイスをカバーする - 繰り返して、2.2を繰り返します。 </li><li> approximat毎日2.5 - 繰り返して、2.2ステップ5日間連続してE 24時間間隔。 </li><li>五日目、治療後に両耳から前庭感覚上皮（3クリステと1卵形黄斑）を抽出（下記のセクション3を参照してください）。 </li></ol> 3.組織抽出20 <ol><li> 26のNaHCO 3、11グルコース、250グリセロール、2.5のKCl、1.2のNaH 2 PO 4、1.2のMgCl 2、および2.5のCaCl 2：（mm）をからなるグリセロールベースの人工脳脊髄液（ACSF）の300ミリリットルを準備します。 7.4のpHを確立し、カルシウム沈殿（曇り）を避けるためにカルボゲン溶液（95％O 2および5％CO 2）、CaCl 2を添加する前にガス。氷スラリーが形成されるように45分間、-80℃の冷凍庫に溶液を冷却する。 </li><li>標識されたスクリュートップマイクロチューブ内のRNA単離、溶解緩衝液を調製する（液体窒素中で凍結および融解の間時の管の圧力変化蓋のポッピングを停止する）に従って製造業者の説明書または通常の実験室の実践に。アルミデュワーフラスコ内の液体窒素をご用意ください。 注：液体窒素を取り扱う際、防護服とeyewareを使用してください。その気体の形態のボリュームが液体の形の約700倍であると窒息の原因となりますので、液体窒素は風通しのよい部屋で使用されていることを確認。 </li><li>深く腹腔内注射を介してケタミン（400 mg / kgを）をマウスに麻酔し。後肢反射が完全にマウスが完全に麻酔をしていることを示すものとして沈静化することを許可する。 </li><li>鋭いステンレス製のはさみでマウスを斬首し、かみそりの刃（丸みを帯びた＃22）を使用して、頭蓋骨の矢状皮膚に沿って切開する。この時点で、全体を通して3.5のステップ- 3.9、組織上の氷冷ACSFの通常のアプリケーションによって、できるだけ涼しい頭蓋冠、脳、および基礎となる前庭装置を保つ。 </li><li> Smalのを作る標準パターンハサミの尖った腕の使い方Lラムダで頭蓋骨を切開し、矢状縫合に沿って切断。 </li><li>静かに脳をドラッグすることなく、骨の下面にできるだけ近いブレードを保ち頭頂骨の下の浅い骨鉗子の一方のアームをスライドさせます。安全で脳が露出するまで後方に横方向に頭頂骨と後頭骨を引き離す。 </li><li>小さなステンレス製のへらを使用して、内耳神経（CN VIII）を露出するために離れて、前と中頭蓋窩から脳を持ち上げる。直接前庭有毛細胞を神経支配する一次求心性軸索上の不必要な緊張を防ぐために神経を横断する。 </li><li> CN VIIIの切断後TOTOで脳を削除します。 </li><li>中頭蓋窩における蝸牛及び末梢前庭器官を含む骨迷路を観察します。各骨迷路の横に二つの小さな切開部を作成し、前方の三半規管を保持し、後で引いて、構造全体を切除同盟国。 </li><li>継続的にカルボゲンで灌流しながら、すぐに（ ステップ3.1で説明したように）の氷冷ACSF溶液を含む解剖皿に切除された迷路を浸す。 </li><li>実体顕微鏡下では、蝸牛によって迷路を押しながら、ピンセットで皿の底に固定します。 <ol><li>前方半規管（SSC）膨大部上記の骨に小さな開口部に傷を付けまっすぐ細かい鉗子を使用してください。 </li><li>静かにすぐに骨の下に到達し、外側に離れて膨大部からフリックすることで、この開口部を拡大する。開口部に鉗子を押し、以下の脆弱な膜迷路を損傷しないためにここに注意が必要です。卵形、前方および横方向の膨大部がすべて露出するまで、このように続けます。可能であれば、また事後膨大部を取り外します。 </li></ol></li><li>細かい鉗子を使用して、静かに卵形嚢を持ち上げ、彼らが完全に切り離されるまで、骨迷路から離れて膨大部。可能な場合には、それらを保持するそれらに関連する半規管によって感覚上皮の損傷を避けるために。いくつかのケースでは、半円形膜状管の近位部は、骨から膨大部を解放するために虹彩鋏で切断する必要があるかもしれない。 </li><li>確実にステップ3.2で先に作成した溶解バッファに鉗子の先端と場所との間に前庭器官を把握する。静かに前庭器官が鉗子から切り離されていることにバッファ内の周りに鉗子を旋回。これは実体顕微鏡下でピンセットを持って来ることでケースです確認してください。 <ol><li>微小管の蓋のネジと、すぐに液体窒素でサンプルを凍結する。 </li></ol></li></ol> 4. RNA抽出およびRT-PCR <ol><li>メーカーの指示又は好ましい実験室プロトコルに従ってメッセンジャーRNA（mRNA）の抽出の標準的な方法に従ってください。 注：mRNAの小さな収量を特定するためにキャリアRNAを利用し、市販のキットが、この中で使用されたプロトコル。低い溶出量は、mRNAの最終濃度を増加させることができる。 注：負の「酵素なし」のコントロール（NECS）と「無DNAテンプレート」コントロール（NTCの）も観察された影響の妥当性を確保するために完了する必要があります。 </li><li>標的遺伝子21-23の相補的DNA（cDNA）の増幅のmRNAの逆転写の標準的な方法を適用する。 </li></ol>

<ol>
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Role of peroxidized cardiolipin and beneficial effect of melatonin.</a> Journal of bioenergetics and biomembranes. 45, 431-440 (2013).</li><li>Zhu, H., Sun, A., Zou, Y., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+metabolic+adaptation+promotes+mesenchymal+stem+cell+therapy+for+ischemia:+a+hypoxia-induced+and+glycogen-based+energy+prestorage+strategy.">Inducible metabolic adaptation promotes mesenchymal stem cell therapy for ischemia: a hypoxia-induced and glycogen-based energy prestorage strategy.</a> Arteriosclerosis, thrombosis, and vascular biology. 34, 870-876 (2014).</li></ol>

著者は、彼らが競合する経済的利益を持っていないと宣言。

ここで説明する代表的な結果は、NIR光の短い頭蓋送達（5日間90秒/日）は、偽処置マウスと比較して老齢マウスにおける抗酸化物質の発現のレベルを上昇させるのに十分であることを示している。放出されながら熱は、ラットの前庭求心24について報告されているよう、ミトコンドリアおよび/ ​​またはニューロンの活性化の源を表すことができる- NIrをすることによって放出される?...

定率性能およびその後滝一般的であり、残念ながら、多くの場合、自然老化1の特徴を定義する。この減少の影響は、物理的および社会的の両方である、と大幅に高齢者の生活の質を低減することができます。応答では、物理療法やリハビリテーションは、滝の研究の焦点となっているが、繰り返し滝の有病率の一貫した減少と関連していない。同時に、作業は、末梢または中枢前庭系（バランスを維持する責任システム）乏しく、これらのシステムを標的とする潜在的な治療戦略の変更や制限された不均衡の根本的な原因を調査する。 加齢性黄斑変性症2-4、アルツハイマー病モデル5-8、およびパーキンソン病9-12を含む加齢関連神経変性疾患に関する最近の研究は、済州の神経保護効果を示した近赤外（NIR）光のmple非侵襲的アプリケーション。さらに、前庭系に、NIrをインビトロ13前庭一次求心性ニューロンの活性を増大させるために使用されてきた。 NIR光のメカニズムはよく理解されていないが、NIrをを使用してほとんどの研究は、NIRは、細胞代謝を促進するために、ミトコンドリア複合体IV（シトクロムcオキシダーゼ）14-17を刺激することを示唆している。前庭感覚上皮ではI型有毛細胞の表皮下プレートは、ミトコンドリア18で緻密であり、そのように治療のNIR治療のための作用部位を表すことができる。 ここで、頭蓋の簡単な非侵襲的処置レジメンは、細胞の代謝を測定するために使用することができる（および含意、ミトコンドリア機能によって）マウス前庭感覚上皮に記載されているNIrを適用した。また議論前庭感覚上皮の調製物であり、それは、NIRはubiquitoの発現を増加させることが示されている感覚上皮における我々の抗酸化剤（スーパーオキシドジスムターゼ1） -以前に蝸牛の有毛細胞の生存19にとって重要であることが示される。

<table border="0" cellpadding="0" cellspacing="0" width="603">
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			<td height="63" style="height:63px;width:216px;">Ketamine</td>
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			<td height="21" style="height:21px;">Standard Pattern Scissors</td>
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			<td style="width:119px;">11252-00</td>
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			<td height="21" style="height:21px;width:216px;">Isolate II RNA Micro Kit</td>
			<td style="width:101px;">Bioline</td>
			<td style="width:119px;">BIO-52075</td>
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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

加齢とのバランス機能の低下を減衰させるための戦略は、主にバランスの作業や運動などの物理的な治療法に焦点を当てている。しかし、これらのアプローチは、バランスの衰退の根本的な原因に対処していない。マウスを用いて、前庭感覚上皮における細胞の代謝に近赤外光（NIR）の影響を評価した。収集されたデータは、このシンプルで安全な介入は自然老化の有害な影響からこれらの脆弱な細胞を保護することができることを示している。 mRNAは、孤立した末梢前庭感覚上皮（膨大部稜と卵形黄斑）から抽出し、続いてcDNAライブラリーに転写した。このライブラリは、その後ユビキタス酸化防止剤（SOD-1）の発現についてプローブした。抗酸化遺伝子の発現は、その後、細胞代謝を定量した。若い（4週間）でのNIR経頭蓋配信を使用し、古い（8から9ヶ月）マウス、および5日間の簡単な治療レジメン（90秒/日AYS）、この作業は、単独NIrを前庭感覚上皮におけるミトコンドリアの機能を改善するのに十分であり得ることを示唆する。前庭有毛細胞の機能を改善するための治療に使用可能な、手頃な価格の、非侵襲的な方法は現在存在しないので、外部NIrを放射線の適用は、前庭感覚上皮inthe細胞代謝の老化の影響に対抗する潜在的な戦略を提供する。

若い（4週間）と古いでNIrを治療の影響を比較する（8から9ヶ月）マウスを、我々は若いた（n = 16）に、酸化防止剤、スーパーオキシドジスムターゼ1（SOD-1）の発現を測定し、古いた（n = 20）偽処理、または近赤外線遮断され、近赤外処理したマウス。 図2は、若い偽処置動物と比較して若いNIrを処理動物で2倍以上のβアクチン正規化されたSOD-1発現の有意な増加を示しているた（p...

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

ミトコンドリア機能障害は、細胞老化の特徴である。本論文では、高齢化マウス前庭感覚上皮におけるミトコンドリア機能を改善するために、非侵襲的近赤外（NIR）処理を使用しています。

近赤外（NIR）の光は、マウス前庭感覚上皮におけるミトコンドリア機能のマーカーの発現を増加させる

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 ビュー.  University of Sydney. ミトコンドリア機能障害は、細胞老化の特徴である。本論文では、高齢化マウス前庭感覚上皮におけるミトコンドリア機能を改善するために、非侵襲的近赤外（NIR）処理を使用しています。 

ビデオ: 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.

近赤外分光法で赤ちゃん、子供と大人の認知機能を探る

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

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.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

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.

ハイC：ゲノムの三次元構造を研究する方法。

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.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

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.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

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.

プラントの脈管構造の三次元構造と機能を可視化するために、高分解能CTを用いた

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.

ヒトiPS細胞のマイクロRNA発現プロファイル、IPから派生網膜色素上皮、および胎児の網膜色素上皮

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.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</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.

ミトコンドリアの構造と機能の研究に<em&gt;ショウジョウバエ</em&gt;卵巣

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.

マウス網膜色素上皮からの一次電池文化

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