我々は感謝して我々の研究室とサポートと有益な議論のための高齢化と栄養（CISBAN）の統合システム生物学センターのすべてのメンバーを承認します。本研究では、バイオテクノロジー·生物科学研究会議（BBSRC）（BB/C008200/1）やウェルカム·トラスト（075294、093088）によりサポートされていました。

<ol>
	<li>Addinall, S. G., Holstein, E., Lawless, C., Yu, M., Chapman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Fitness+Analysis+shows+that+NMD+proteins+and+many+other+protein+complexes+suppress+or+enhance+distinct+telomere+cap+defects.">Quantitative Fitness Analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects.</a> PLoS Genetics. 7 (4), e1001362 (2011).</li><li>Addinall, S. G., Downey, M., Yu, M., Zubko, M. K., Dewar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomewide+suppressor+and+enhancer+analysis+of+cdc13-1+reveals+varied+cellular+processes+influencing+telomere+capping+in+Saccharomyces+cerevisiae.">A genomewide suppressor and enhancer analysis of cdc13-1 reveals varied cellular processes influencing telomere capping in Saccharomyces cerevisiae.</a> Genetics. 180, 2251-2266 (2008).</li><li>Lawless, C., Wilkinson, D., Young, A., Addinall, S., Lydall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonyzer:+automated+quantification+of+micro-organism+growth+characteristics+on+solid+agar.">Colonyzer: automated quantification of micro-organism growth characteristics on solid agar.</a> BMC Bioinformatics. 11, 287-28 (2010).</li><li>Tong, A. H., Evangelista, M., Parsons, A. B., Xu, H., Bader, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+genetic+analysis+with+ordered+arrays+of+yeast+deletion+mutants.">Systematic genetic analysis with ordered arrays of yeast deletion mutants.</a> Science. 294, 2364-2368 (2001).</li><li>Costanzo, M., Baryshnikova, A., Bellay, J., Kim, Y., Spear, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+landscape+of+a+cell.">The genetic landscape of a cell.</a> Science. 327, 425-431 (2010).</li><li>Blomberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+growth+rate+in+high-throughput+growth+phenotyping.">Measuring growth rate in high-throughput growth phenotyping.</a> Curr. Opin. Biotechnol. 22 (1), 94-102 (2011).</li><li>Maringele, L., Lydall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXO1-dependent+single-stranded+DNA+at+telomeres+activates+subsets+of+DNA+damage+and+spindle+checkpoint+pathways+in+budding+yeast+yku70&#916;mutants.">EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70&#916;mutants.</a> Genes and Dev. 16 (15), 1919-1933 (2002).</li><li>Warringer, J., Blomberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+in+environmental+arrays+allows+analysis+of+quantitative+phenotypic+profiles+in+Saccharomyces+cerevisiae.">Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae.</a> Yeast. 20, 53-67 (2003).</li><li>Hillenmeyer, M., Fung, E., Wildenhain, J., Pierce, S., Hoon, S., Lee, W., Proctor, M., St. Onge, R., Tyers, M., Koller, D., Altman, R., Davis, R., Nislow, C., Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+genomic+portrait+of+yeast:+uncovering+a+phenotype+for+all+genes.">The chemical genomic portrait of yeast: uncovering a phenotype for all genes.</a> Science. 320 (5874), 362-365 (2008).</li><li>Smith, A. M., Durbic, T., Oh, J., Urbanus, M., Proctor, M., Heisler, L. E., Giaever, G., Nislow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+Genomic+Screens+of+Barcoded+Yeast+Libraries.">Competitive Genomic Screens of Barcoded Yeast Libraries.</a> J. Vis. Exp. (54), e2864 (2011).</li><li>Lederberg, J., Lederberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replica+plating+and+indirect+selection+of+bacterial+mutants.">Replica plating and indirect selection of bacterial mutants.</a> J. Bacteriol. 63, 399-406 (1952).</li><li>Queller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+ecology:+The+social+side+of+wild+yeast.">Behavioural ecology: The social side of wild yeast.</a> Nature. 456, 589-590 (2008).</li></ol>

利害の衝突が宣言されません。

文化の希釈系列のスポットテスト9、通気液体培養9およびレプリカ平板13の成長曲線の決定：QFAは、多くの感覚に3つの確立されたベンチスケールの微生物学的技術の直接の子孫である。これらの3つの方法は、要約し、QFA及び表2の他のハイスループット技術と比較されます。希釈系列のスポットテストでは、接種希釈の範囲から成長した文化のシリーズのコロニーを形成する能力とひずみの適合性を定義するのに対し、単一文化の繰り返し観測によるQFA対策ひずみフィットネスは、成長曲線を構築する。単一文化の適合性を定量化すると、より多くの株が同一の条件下では、同時に検査することができます。異なる環境や遺伝的背景の培養フィットネスの違いをテストするときに文化の複製配列は、最も有用菌株コレクションの繰り返しテストすることができます。 QFAプロトコルは、提示ここで、独立した文化（ロボットQFA、 表2）数千人の成長を観察するための適切なインキュベーションとイメージ寒天プレートを複製接種するために高価なロボット装置を使用しています。 QFAが確立し、伝統的なラボの技術に基づいているので、しかし、それはまた、手動の手順（マニュアルQFA、 表2）ロボットの援助を置き換えることによって、はるかに安価に行うことができます。マニュアルQFAは、手動でピンツールとおよび写真撮影のためのインキュベーターからプレートの手動転送を使用して、寒天プレート上に培養物の複製および接種が含まれます。同じ計算解析ワークフローのいずれか実験的なデザインから成長曲線を生成するために適用することができます。 QFAフィットネスの見積もりが比較的正確であるように見える。 図4では、機能的に関連する遺伝子欠失の4例では、クラスタの平均適応度が強調表示されます。 2の自主的にそれぞれの機能的関連遺伝子クラスのメンバーの近接ENT遺伝的背景は、（ura3ΔとCDCl 3-1）QFAフィットネス推定値の再現性を示しています。たとえば、わずか3遺伝子欠失（アウト可能な4300の）保存されたMRX複合体の3つのメンバーを分離します。 微生物株の増殖特性をテストするためのQFAに高スループットの選択肢がバーコードライブラリ11,12および他のより完全に8を 表2にまとめて記載されている分光光度プレートリーダー10の光学濃度の動態をキャプチャ画面でSGA 5,6、競争画面を含む。培養は固体培地の表面上に成長QFAとSGAプレート（寒天ベースの方法、 表2）ロボットが迅速かつ容易に処理することができます。固体寒天表面の文化はよく成長全体に通気されており、細胞は社交的な微生物がコミュニティ14に成長することができ、固定の文化で成長することができます。左そのまま、微生物群集独自のマイクロ環境では、エタノールなどを分泌する毒素をffect、おそらく細胞間シグナル伝達。しかし、十分な通気を達成するために必要な、液体培養の連続混合、人工的に成長のそのモードに影響を与える可能性微生物群集とその微小環境を破壊する。文化間の細胞を運ぶ液体の飛沫の少ないチャンスがあるので、クロスコンタミネーションは、固形寒天方法の懸念が少ないです。汚染が固体寒天アッセイにおける外国空気を媒介とする微生物によって発生した場合、それはしばしば固体寒天プレートの目視検査によって検出され、占めまたは削除することができます。 QFAのスループットは、2つの方法で並列液体の方法に比べて有意に高くなっています。まず第一に、QFA（およびSGA）プレート上に接種した培養物は、プレート当たり複数の独立した文化を与え、より密に一緒にパックされています。 QFAで308の文化は、非実験的なエッジの培養（ 図1）COMはカウントしません、一般的に存在するプレート96または100文化と液体培養を並列に比較されます。第二に、QFA実験は、プレートのはるかに大きな数に拡張することができます。シングルQFA実験で分析したプレートの数は、インキュベーターまたは暖かい部屋、最小許容画像キャプチャの周波数と達成可能な最大キャプチャ周波数で使用可能なスペースによって制限される一方、液体増殖実験ではプレートの数を強く制限されているプレートリーダー（通常は1つまたは2つのプレート）、またはプレートリーダー（通常は25から50プレート）に接続されているスタッカの容量による。我々は最近、37884同時の文化を与え、123プレート、308実験的な文化とそれぞれの完全に自動化されたQFA実験を行った。我々は、液体中で成長して独立した文化の達成可能な最大数は96（文化/プレート）×50（プレート/スタッカ）私たちのQFAのスループットに比べ約10倍未満である= 4800であると推定している。液体培養の画面の代わりに、多くの自動化されたGROを利用することである並行してWTHデバイス（Blombergの8による評価から表1）、1つまたは2つのプレートの容量を持っている各。各デバイスの温度制御は独立している、などの条件が同一ではありませんが、あると仮定し、このワークフローでは、少なくとも190のようなデバイスは、QFAのスループットを（デバイスあたり200液体培養と仮定して）一致するように必要になります。 要約すると、QFAは有用小規模焦点を当てた実験や、高スループットのスクリーンの両方で、量的成長の表現型を収集するために適用することができ、高品質なワークフローです。それは、ロボット装置の要件を変えることで効果的に適用されるのに十分な柔軟性があります。 QFAワークフローの計算コンポーネントは、自由に利用できる、オープンソースコードに基づいています。

<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>レプリカプラター</td><td>シグマ</td><td> R-2508 </td><td> 1/8 &quot;径のピンと96ピンマニュアルpintool </td></tr><tr><td>ミックスメイト</td><td>エッペンドルフ</td><td> 5353 000.014 </td><td></td></tr><tr><td> Biomek FX </td><td>ベックマン</td><td> A31842 </td><td></td></tr><tr><td> Teleshake </td><td>サーモフィッシャーサイエンティフィック</td><td> 50095890 </td><td> Biomek FXにインストールされている</td></tr><tr><td> BM3-SC </td><td> S＆Pロボット株式会社</td><td> BM3-SC </td><td> 192棚回転カルーセル</td></tr><tr><td> spImager </td><td> S＆Pロボット株式会社</td><td> spImager </td><td>高解像度マニュアルイメージング</td></tr><tr><td> CytomとspImagerに</td><td> S＆Pロボット株式会社</td><td>カスタム</td><td>温度が自動化された高分解能イメージングを制御</td></tr><tr><td>熱交換器とCytomat 6001 </td><td>サーモフィッシャーサイエンティフィック</td><td> 51022222 </td><td> spImagerに接続されている</td></tr><tr><td> ECOLINE RE207 </td><td>ラウダ</td><td> RE207 </td><td> Cytomatに接続されている</td></tr><tr><td>カルーセルとspImager </td><td> S＆Pロボット株式会社</td><td>カスタム</td><td>自動化された高分解能イメージング</td></tr><tr><td> FP12pinsのロボットピンツールフィクスチャ</td><td> V＆Pサイエンティフィック</td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96×FP12ピン</td><td> V＆Pサイエンティフィック</td><td> FP12 </td><td>長い50.4ミリメートル、17ミリメートル露出したピンの長さ</td></tr><tr><td>ピンツールのドッキングステーション</td><td> V＆Pサイエンティフィック</td><td> VP425 </td><td>ドッキングステーションベースはピンのファンの乾燥を可能にするために除去</td></tr><tr><td>ピンのクリーニングブラシ</td><td> V＆Pサイエンティフィック</td><td> VP425 </td><td> N / A </td></tr><tr><td>レプリカプラター</td><td>シグマ</td><td> R-2508 </td><td>マニュアルのピンツール</td></tr><tr><td>蓋ヌンクOmniTray </td><td>ヌンク</td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96ウェル滅菌ポリスチレンプレート</td><td>グライナーBioOne </td><td> 655161 </td><td> N / A </td></tr><tr><td> 96ウェル滅菌ポリスチレン蓋</td><td>グライナーBioOne </td><td> 656171 </td><td> N / A </td></tr></tbody></table> 表3。特異的試薬および装置。

a quantitative fitness analysis workflow

定量的なフィットネスの分析（QFA）は、並列1,2,3,4で育った微生物培養の適応度を比較するための実験と計算のワークフローです。 QFAは、単一の文化の焦点を絞っ観測に適用されるが、独立した文化の数千までの調査のゲノムワイドな遺伝的相互作用や薬物スクリーンのための最も有用であることができます。中央の実験方法では、インキュベートし、定期的に撮影されている固体寒天プレート上に独立した、希釈液を微生物培養の接種です。各時間ポイントからの写真は、定量的なフィットネスの措置が導出できるようになり、成長曲線を構築するために使用されている定量的な細胞密度の見積もりを、製造、分析されます。文化適応度は遺伝的相互作用の強さや薬剤感受性を定量化し、ランク付けするのに比較することができます。すべての治療の培養適応に与える影響は、基板の寒天（例えば、小分子、抗生物質や栄養素）に追加したり、外部のプレートに適用LY（例えばUV照射、温度）がQFAによって定量化することができます。 QFAワークフローでは、成長率は96ウェルまたは200ウェルプレートリーダーで並列液体培養の分光光度測定により得られるものに類似して推定し生成します。重要なのは、QFAは、このような方法と比較して有意に高いスループットを持っています。 QFA培養は固体培地の表面に成長するので、よく撹拌又は振盪を必要とせず、成長中に通気されています。 QFAのスループットは、いくつかの合成遺伝子アレイ（SGA）のスクリーニング方法5,6のそれと同じくらい高くはありません。 QFAの文化が頻繁に寒天に接種される前に希釈されているのでしかし、QFAは3指数と彩度の段階を含む、より完全な成長曲線を、キャプチャすることができます。たとえば、成長曲線の観測は、以前は1説明したように文化の倍加時間は、高精度を直接推定することができます。 ここでは提示S.数千人に適用される特定のQFAプロトコル自動的に接種、培養し、撮像中にロボットによって処理される酵母の培養。これらの自動化のいずれかの手順は、スループットに関連して減少し、同等の、マニュアルの手順に置き換えられ、我々はまた、低いスループット·マニュアル·プロトコルを提示することができます。同じQFAソフトウェアツールのいずれかのワークフローで撮影した画像に適用することができます。 私たちは出芽酵母の培養にQFAを適用する豊富な経験を持っているS.酵母しかし、我々はQFAは分裂酵母S.の文化を調べるために均等に役立つことを期待酵母と細菌培養。

Watch this Scientific Journal Video about A Quantitative Fitness Analysis Workflow at JoVE.com

A Quantitative Fitness Analysis Workflow

定量的なフィットネスの分析（QFA）は、微生物の培養適応度を推定するための実験と計算手法の相補的なシリーズです。 QFAは、遺伝的変異、薬剤や微生物の成長の他の適用された治療の効果を推定します。単一の文化の焦点を絞っ分析から並列文化の何千もの実験のスケーリングを設計することができる。

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. ...

Research

JoVE Journal

Biology

14.3K ビュー.  Newcastle University Medical School. 定量的なフィットネスの分析（QFA）は、微生物の培養適応度を推定するための実験と計算手法の相補的なシリーズです。 QFAは、遺伝的変異、薬剤や微生物の成長の他の適用された治療の効果を推定します。単一の文化の焦点を絞っ分析から並列文化の何千もの実験のスケーリングを設計することができる。

ビデオ: A Quantitative Fitness Analysis Workflow

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸の定量的なPhosphoproteomicsは刺激を受けてサッカロマイセスセレビシエ</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

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 Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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 ...

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 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 ...

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 male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

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 ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

ヒストンの抽出からデータ解析まで：ボトムアップ質量分析法を用いてヒストンの翻訳後修飾の解析のための完全なワークフロー

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...