We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

倫理声明：動物に関わるすべての研究は施設のIACUCによって承認されなければならない（以下のデータについては、プロトコルは、モンテフィオーレIACUC、プロトコル＃11-6-240と＃13-7-100によって承認された）。撮影時まだそれらを維持する唯一の目的のために示されているように、動物が軽い麻酔を提供され、このプロトコルに必要ない痛みを伴う手続きはありません。 写真の1.取得<ol><li>印刷されたテキストと紙を用いたゲルイメージャのためのビューのフォーカスとフィールドを設定します。撮影した地域全体の光源の均一性を確認してください。均一性を確保するために、反射写真撮影のための光源を内蔵したゲルイメージャを使用しています。これはさらにグレースケールの分析には適していない動物のシルエットを作成すると同じように、透照光源を使用しないでください。 注：これは関心領域（ROI）との意志を越え、光の平均化を提供する、やや焦点の外にマウスを置きます興味のある非常に小さい（ すなわち、&lt;10ピクセル）の領域について量子的エラーを減らすのに役立ちます。関心の大きな領域は、このマイナーな焦点調節によって影響されず、またそれらは、動物の大きさの違いによって影響される。 </li><li>これは急速な麻酔効果および迅速な回復を提供し、複数の動物を撮影するために最適であるように、：ケタミン（100mg / mlの）/キシラジン（20 mg / ml）で（1 2）を使用して動物を麻酔。 注記：動物が静止画撮影を可能にするのに十分であるときに麻酔が確認された。動物は一般的に10〜15分以内にこの光麻酔から回復したように、獣医師は眼軟膏の使用をお勧めしていません。 </li><li> （できるだけ平行に近い）の垂直配向ゲルイメージャーで麻酔した動物を置きます。 <ol><li>背写真の場合は、延長手足と腹臥位で動物を配置。 </li><li>腹写真のために、動物が横方向に回転しないように注意しながら、仰臥位で動物を配置。 </li&gt; </ol></li><li>撮影した領域でのグレースケール標準を置きます。 </li><li>閉じるアクセスドア。重要：周囲光露光の変化を導入することができる。 </li><li>取得の直線範囲内の関心領域を配置露光Fストップを設定する（Fストップを読み取る）。 注：画像が飽和している場所ほとんどのシステムが表示されます。 </li><li>写真を撮る。 </li><li>変更標準および関心領域の両方を取得（参照の直線範囲内に留まるように、1からFストップを増加または減少させることによって、直線範囲内の関心領域を配置し、別の露出設定にFストップF-停止）。 </li><li>写真を撮る。 </li><li>撮影が完了すると、加温テーブルに動物を配置し、それらが伏臥を維持できるようになるまで監視。ケージに動物を返します。一度、すべての動物が完全に回復している動物施設への動物のグループを返します。 </li></ol>光の吸収2.定量&lt;/ pの&gt; <ol><li>使用した動物の画像上で関心のマーク領域は、ゲルイメージャのためのソフトウェアを提供した。 <ol><li>全動物の背ビューについては、可能な限り横方向に限り延長、下肢への上肢から延びる長方形または楕円形のイメージを使用するように、 図1Aに示すように、箱のいかなる部分も、動物の背中を越えて延びていない。 NOTE：一つは、フリーハンド描画ツールを使用して、関心のある領域をマークできた。 </li><li>骨盤領域をカバーする1と図1（b）に示すように胸のエリアをカバーする1：全体の動物腹ビューについては、2の長方形を使用しています。 </li><li>必要に応じて、関心、 すなわち、薬剤投与の位置、マークのより小さな領域のために。 </li></ol></li><li>グレースケール吸収規格上の関心のマーク領域（複数可）。 </li><li>関心のマークされた領域のそれぞれからレコード吸収。 </li></ol> 3.分析吸収レベルobtaine関心領域の研究開発は、写真間の比較のために、バックグラウンドの標準に正規化する必要があるかもしれない。吸収への曝露の関係は両対数（ すなわち、関係がログ（曝露の間に線形である）とログ（吸収））である。この関係を用いて、関心領域の吸収は、吸収が異なる時点（試験プロトコル内、すなわち、連続測定）で撮影されたものを含む別の写真の間で直接比較することを可能にする標準的なバックグラウンド値に正規化することができる。これらの補正を実行するための手順は、オプションのステップ3.1および3.2に詳述されている。 <ol><li>グレースケールの吸収標準から得られた値を使用して、ログ対ログの（オプション）プロット曲線（露出）（吸収）。 </li><li>次のように（オプション）各写真の標準の変動を調整します。 <ol><li>秒で決定されるように（参照のためおよびFストップ（ステップ1.6で決定されるように）読み取るためFストップを選択1.8）をTEP。 </li><li>読んでFストップは全く写真に標準の平均吸収を計算します。この平均値は参考基準値（RSV）である。 </li><li> （デルタ-ROI-1、デルタ-ROI-2 ... ..delta-ROI-X）規格（デルタ-S）のために、各定義されたROIのためにそれぞれの写真に写っている読書と参照F-ストップ設定間の吸収の差を計算する</li><li> *（デルタROI-X / - F-ストップ+（F-ストップを読んで、標準の吸収RSV）を読んで補正吸収（ROI-X）=吸収（ROI-X）：次のように各ROIに対する補正吸収を計算しますデルタS） </li></ol></li><li>実験データをコンパイルし、連続変数（ すなわち、ANOVA、T検定）のための標準的な統計的手法を用いてデータ解析を行う。 </li></ol>

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Authors have nothing to disclose.

本報告書では詳細な説明は、げっ歯類で脱毛を定量化するための新しい技術の提供されています。この技術は、ほとんどの実験室で容易に利用可能である画像取得と分析のためのゲルイメージャ、機器を利用する。測定は、技術（ 図4から5）における小さな変化に対してロバストであることが示されており、治療モデルにおける視覚脱毛（ 図6）の程度によく相関し?...

脱毛（抜け毛）は複数の原因と心理的、感情的苦痛イベントになることができます。男性型脱毛症は35歳1によって男性の約2/3に影響を及ぼし、脱毛症の最も一般的な原因である。抜け毛の同様のパターンは、多嚢胞性卵巣症候群の女性において観察することができる。これらの疾患の両方において、脱毛はアンドロゲン媒介性である。脱毛症はまた、人口2の1.7％に影響を与える自己免疫疾患、円形脱毛症、として発生する可能性があります。脱毛症は、化学療法3のようないくつかの医学的治療の副作用として発生する可能性があります。化学療法患者の割合が高い（65から85までパーセント）は、脱毛症4,5ある程度の経験する。脱毛の心理的な影響は十分に化学療法の設定で研究されている。化学療法誘発性脱毛症、不安、抑う ​​つ、負の身体イメージをもたらすことができ、自尊心と6,7-幸福の減少感を低下させた。高いPE女性のがん患者のrcentage（百分の47から58）は脱毛は、化学療法の最も外傷側面であると考えて、そして脱毛4,6の恐れのための最大8％の下落治療。脱毛8,9の心理的にも医学的結果を減少させるための治療をサポートするための男性ホルモン性脱毛症における証拠もある。同様に、円形脱毛症は、重度の心理的な影響2、及び脱毛の斑点状の性質が脱毛の他のほとんどの原因はより不快化粧結果につながることが報告されている。 穏やかな抗アンドロゲン作用（ すなわち、スピロノラクトン）を有する薬剤が脱毛症の治療などの限られた成功を収めて使用されていたが、脱毛症のための最初の効果的な薬物療法は、ミノキシジル10だった。この血圧降下は、毛髪の成長を引き起こすの観察された副作用を有しており、現在脱毛症の多くの形態のための局所療法として使用される。しかし、応答はいくつかの科目だけ遅く表示で、しばしば不完全である脱毛ではなく、実際の再成長の10る。フィナステリドは、部分的な全身のアンドロゲン遮断を犠牲にしてアンドロゲン性脱毛症の改善で、その結果、ジヒドロテストステロンのII5α還元酵素をブロック変換を入力する競合的アンタゴニストである。長期的（10年）療法との回答率は50％前後11である。全体として、この分野におけるかなりの研究にもかかわらず、脱毛のための適切な治療は依然として存在しない。 何十年もの間、科学者や臨床医は、臨床試験で頭皮毛髪の成長を測定する方法を検討した。脱毛症を治療する薬剤の開発により、毛髪の成長と、治療に対する特異的応答を測定する、信頼性の高い経済的な低侵襲手段に対する大きな必要性があった。脱毛障害を有する患者における毛髪密度の正確な定量化のための画像解析技術がtechniquの様々な方法を用いて、過去に一貫して有効な結果が得られた定義された頭皮領域14に毛量を定量化するためのデジタル画像12の解析、個々の毛髪及び皮膚病変13、および顕微鏡走査の画像解析を含むエス。 上記の方法論は、臨床試験における発毛促進介入のための有効性の改善された評価を提供している残念ながら、これらの方法は、前臨床研究において、げっ歯類の研究に適用されていない。我々の目的は、研究者は、より正確に脱毛症の種々の形態のための新たな治療アプローチを評価し、比較することを可能にするであろう、マウスにおいて、脱毛を定量化するために一貫して信頼性の高い方法を開発することである。私たちは、茶色や黒い髪を持つマウスにおける毛髪密度の迅速かつ信頼性の高い定量化が可能になりますほとんどの実験室で容易に利用可能な機器を使用して方法論を開発した。この方法はwaxinから化学療法誘発性脱毛症、円形脱毛症、および脱毛症のマウスモデルにおいて試験されているグラム。詳細なプロトコルは、マウスのC57BL / 6及びC3H / HEJ株からの検証データを含む、これらの測定を実行するために提示されている。この技術は、毛幹中での顔料の光吸収を検出することに依存しているように、白マウス又はアルビノマウスにおける発毛を検出するために使用することができない。

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

脱毛症は、男性型脱毛、多嚢胞性卵巣症候群、および円形脱毛症など、さまざまな条件で発生する可能性が脱毛の一般的な形式です。脱毛症は、癌患者における化学療法の副作用として生じ得る。本研究では、私たちの目標は、研究者が正確に評価し、脱毛症のこれらの様々な形態のための新たな治療アプローチを比較することができるようになりますマウスでの脱毛を、定量化するための一貫性と信頼性の高い方法を開発することであった。この方法は、マウスに黒（または灰色）の毛髪の量にラフ比例して発生する光吸収を測定する、マウスの画像を取得し、処理するための標準的なゲルイメージャを利用しています。このようにして定量化されたデータは、その後、標準的な統計的手法（ すなわち、ANOVA、T検定）を用いて分析することができる。この方法は、ワックスの化学療法誘発性脱毛症、円形脱毛症および脱毛症のマウスモデルにおいて試験した。このレポートでは、詳細なプロトコルは、PRESですマウスのC57BL / 6及びC3H / HEJ株からの検証データを含む、これらの測定を行うために取得済み。この新技術は、相対アプリケーションのシンプルさ、ほとんどの研究室で容易に利用可能である機器への依存、そして客観、主観的な評価よりも堅牢である定量的な評価を適用するなど、多くの利点を提供しています。マウスにおける毛髪成長の定量化の改善は、脱毛症モデルの研究を改善し、前臨床試験で有望な新しい治療法の評価を容易にする。

この技術は、C3H / HEJ生着マウス、16円形脱毛症のマウスモデルを用いて検証した。これらの動物は、時間をかけて徐々に進行するグローバル脱毛を開発する。しかし、脱毛パッチで発生し、かなりの変動を導入し、定性的な評価を妨げ、次の1匹のマウスから異なる場合があります。このモデルは、有効性の評価として、マウスの異なる位置での光学密度の測定値の間の相関をテスト?...

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

グレースケール解析を用いたマウスの抜け毛の定量分析のための新たな手法

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

a-new-technique-for-quantitative-analysis-hair-loss-mice-using

Research

JoVE Journal

Biology

8.9K ビュー.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

ビデオ: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

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.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

生細胞イメージングとの胚上皮細胞の定量分析アフリカツメガエル</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量的なフィットネスの分析ワークフロー

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

高度な3D蛍光顕微鏡を用いたオートファジーの定量分析

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

に基づいた多タンパク質複合体の比較分析のための新たなアプローチ<sup&gt; 15</sup&gt; N代謝標識および定量的質量分析

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

ペプチドエピトープの定量分析のための高スループット、MHC II結合アッセイ

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

水生動物使用グループ固有の rDNA プライマーの遺伝的腸内容分析の研究プロトコル

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...