We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

The authors declare no competing interests.

酵母細胞におけるエンドサイトーシスイベントの定量化のためのpHluorinのアプリケーションは、蛍光タグは、タンパク質（ 図1A）の細胞質尾部に融合された貫通貨物を利用します。ここに記載のアッセイのために、貨物はpHluorinタグがニュートラルな環境にさらされたときに明るく蛍光であるが、pHluorinタグは酸性条件に遭遇したときに急冷になります。したがって、pHluorinタグ付...

小胞輸送は、真核細胞内小器官の同一性および機能を維持する上で重要な役割を果たし、個々の細胞区画のタンパク質および膜組成を調節するための重要なメカニズムです。エンドサイトーシスを介して生成された小胞は、その後のリサイクルまたはリソソームに標的化するための表面から膜およびタンパク質を除去に対し、原形質膜で、エキソサイトーシス小胞の融合は、細胞の表面に新たなタンパク質や膜を実現します。このように、エンドサイトーシスは、栄養摂取のためおよび細胞外環境への応答のために重要です。エキソサイトーシスおよびエンドサイトーシスは、形質膜の表面積を調節するため、および損傷を受けたタンパク質の代謝回転を可能にするようにバランスされています。 酵母および哺乳動物細胞での研究には、専用の様々なことに応答して、エンドサイトーシスに関与するタンパク質の大規模な数、ならびに特定の貨物の内在化を促進する複数のエンドサイトーシス経路またはその行為を同定していますvironmental条件。最もよく研​​究経路は、クラスリンおよびサイトゾルアクセサリータンパク質は、発生期のエンドサイトーシス小胞を安定化させるためにコート構造に集合したクラスリン依存性エンドサイトーシス（CME）、です。出芽酵母サッカロマイセス・セレビシエにおける実験は、力学と多くのエンドサイトーシスタンパク質1-3のための募集のために重要な洞察が得られています。特に、CMEの機械は非常に進化の過程で保存されているような酵母におけるCME関連タンパク質の大部分は人間のオルソログを持っていること。このように、出芽酵母は、高等真核生物に保存されているエンドサイトーシスのメカニズムを理解するための重要なツールとなっています。例えば、出芽酵母を用いた研究では、追加のエンドサイトーシスのアクセサリーの動員に続いて、CME構造（酵母における皮質アクチンパッチと呼ばれる）の形成と成熟がクラスリンと貨物結合アダプタータンパク質で始まる、多くのタンパク質の順次募集を伴う決定しましたタンパク質、ARP2 / 3媒介アクチン重合の活性化、および小胞の切断の1,2に関与するタンパク質の動員。皮質アクチンパッチにCME機械タンパク質の募集は、高度に秩序とステレオタイプのプロセスであり、多くのタンパク質のための募集の正確な順序は、CMEの機械の他の構成要素に関して確立されています。重要なことは、最近の研究では、哺乳動物細胞4でクラスリン被覆ピットでのCMEのタンパク質のための募集の同様の順序を確認しています。 CMEに加えて、多くの細胞型は、原形質膜5-7の小胞形成および内在化を促進するための代替メカニズムに頼る一つ以上のクラスリン非依存性エンドサイトーシス（CIE）の経路を有します。酵母では、我々は最近、低分子量GTPase Rho1とその活性化グアニンヌクレオチド交換因子（GEF）、ROM1 8,9を利用CIE経路を同定しました。 Rho1は、アクチン重合1を促進するためにformin Bni1を活性化させます酵母12でクラスリン非依存性エンドサイトーシスは、このフォームに必要とされる0,11、。また、タンパク質のα-アレスチンファミリーは、CME 13-17を通じて貨物のユビキチン化およびその後の内在化を促進し、またおそらくCIE 18に関与するタンパク質との直接的な相互作用を介して、CIE経路を介して貨物の内在化を促進するために、ユビキチンリガーゼRSP5を募集します。 CIE経路の様々なもRho1オルソログ、RhoAの9,19に依存しているクラスリン依存せず、食作用経路を含めた哺乳動物細胞、中に存在します。哺乳類CIEにおけるRhoAの役割は十分に理解されていません。従って、出芽酵母での研究は、哺乳類のCIEに適用可能な追加の機械的な洞察を提供してもよいです。 エンドサイトーシス事象の正確な定量は、エンドサイトーシスを調節する特定のタンパク質の役割に関する重要な情報を提供することができ、および貨物の内在またはprogressio上の変異の影響を明らかにすることができますエンドサイトーシス経路を介して、n個。この目的のために、生化学的方法は、リガンドの取り込みを監視するために、またはエンドサイトーシス積荷タンパク質の分解の速度を測定するために利用することができます。生細胞において、関心対象の貨物に緑色蛍光タンパク質（GFP）及びその変異体の融合は、貨物輸送の直接可視化を可能にします。 GFPは、（酵母または液胞）リソソームでの分解に耐性があるのでしかし、GFPタグ貨物は、エンドサイトーシスの定量化のための使用が制限されています。また、GFP蛍光は、pHの変化に部分的にのみ敏感であり、液胞ルーメン20,21内で検出可能なままです。その結果、GFPタグの蛍光は、貨物の残りの部分が劣化したずっと後に液胞に固執し、貨物の強度の全細胞ベースの定量化が原因液胞の長期的なGFP蛍光に不正確になることがあります。 液胞GFP蛍光の欠点を克服するために、我々は、以前にsuperecliptic pHを使用しましたluorin、中性pHで明るく蛍光を発するが、そのようなリソソーム液胞/ 20,22,23の内腔などの酸性環境下で蛍光を失うGFPのpH感受性変異体です。エンドサイトーシス貨物の細胞質尾部にpHluorinタグを配置することは、原形質膜でとpHluorinタグが細胞質（ 図1A）に露出したまま初期エンドソーム、上の荷物の可視化を可能にします。初期エンドソームが成熟するにつれ、エンドソームは、多胞体（MVBs）24を生成し 、エンドソームの内腔に出芽小胞への輸送のために必要な複雑な（ESCRT）機械パッケージ表面局在貨物を仕分け。内部MVB小胞に組み込まれている貨物については、pHluorinタグは小胞内腔に向いています。内部MVB小胞が酸性化されています。彼らはMVBs 20に成熟としてこのように、pHluorinタグ付きカーゴは、初期エンドソームの蛍光を失います。液胞とMVBのその後の融合は、MVB腔の内容を提供します分解のために、とpHluorinタグは液胞内腔の酸性環境中で急冷したまま。 本稿では、酵母における細胞質pHluorinタグで貨物を用いた定量的エンドサイトーシスアッセイの詳細な説明を提供します。 pHluorinタグ貨物を発現する株は、特定の貨物の性質および輸送の挙動に応じて、運動及び/又はエンドポイントアッセイに使用することができます。さらに、いくつかのpHluorinタグ貨物は、フローサイトメトリーなどのハイスループット分析法に適しています。重要なことは、pHluorinタグは、野生型と変異株におけるエンドサイトーシス機能のエンドサイトーシスとの比較の定量化を可能にする、生きた細胞内のエンドサイトーシス事象を研究するための汎用性の高いツールです。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adenine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A8626-25G</td>
			<td style="width:167px;">Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>Use for preparation of plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Difco Yeast Nitrogen Base (without amino acids)</td>
			<td>BD</td>
			<td>291920</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concanavalin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C5275-5MG</td>
			<td>Use for coating of chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher</td>
			<td>BP350-1</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td>Fisher</td>
			<td>BP382-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>Acros</td>
			<td>125121000</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine</td>
			<td>Fisher</td>
			<td>BP386-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td>Fisher</td>
			<td>BP388-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Fisher</td>
			<td>BP395-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tyrosine</td>
			<td>Acros</td>
			<td>140641000</td>
			<td>Use for preparation of amino acid mixture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc Lab-Tek Chambered Coverglass (8-well)</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">155411</td>
			<td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axiovert 200 inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Custom Build</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Objective should be 100X, 1.4NA or higher</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sensicam</td>
			<td style="width:71px;">Cooke Corporation</td>
			<td style="width:119px;"></td>
			<td>Camera should have 12-bit or higher dynamic range</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">X-Cite 120PC Q Illumination Source</td>
			<td style="width:71px;">Excelitas Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Slidebook 5 software</td>
			<td style="width:71px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ImageJ software</td>
			<td style="width:71px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

緑色蛍光タンパク質（GFP）およびその変異体は、広くタンパク質の局在や、細胞骨格の再構築と生きた細胞内小胞輸送などのイベントのダイナミクスを研究するためのツールを使用しています。キメラGFP融合物を用いた定量方法は、多くの用途のために開発されています。しかし、GFPは、タンパク質分解に対して多少耐性があるので、その蛍光は、エンドサイトーシス経路に貨物輸送の定量化を妨げる可能性がリソソーム/液胞に持続します。エンドサイトーシスとエンドサイトーシス後の人身売買のイベントを定量化するための別の方法はsuperecliptic pHluorin、酸性環境中で急冷されるGFPのpH感受性の変異体を使用しています。多胞体（MVBs）およびリソソーム/液胞の内腔への配信への貨物の取り込み時に蛍光の減衰における膜貫通貨物タンパク質結果の細胞質尾部にpHluorinのキメラ融合。このように、液胞蛍光の消光はquantifiを容易にエンドサイトーシスとエンドサイトーシス経路における初期の事象のカチオン。本稿では、フローサイトメトリーを用いて、蛍光顕微鏡を介したエンドサイトーシスの定量化のためのpHluorinタグ付き貨物だけでなく、集団ベースのアッセイを使用する方法について説明します。

構成的に内在化エンドサイトーシス貨物タンパク質STE3の定常状態の局在化および定量化 生きている細胞、STE3、酵母におけるA因子フェロモン受容体の細胞質C末端テイルを有するキメラGFPとpHluorin融合の局在化pHluorinタグ貨物はエンドサイトーシスの定量化のために利用できることを実証するために、比較しま...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

applications-phluorin-for-quantitative-kinetic-high-throughput

Research

JoVE Journal

Biology

10.8K ビュー.  The Johns Hopkins University. Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

ビデオ: Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

出芽酵母の複製寿命の測定

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

生物学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

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 ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

シングル出芽酵母細胞での寿命と細胞表現型を測定するためのマイクロ流体デバイスを使用しました

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...