この作品は、NIHのグラント1R21AG031965 - 01A1によってサポートされていました。 MKは、高齢化のエリソン医学財団新奨学生です。

パート1：高齢化の文化の調製<ol><li> YEPD寒天プレート上に凍結ストック（1％酵母エキス、2％バクトペプトン、2％寒天、2％グルコース）から興味のストリーク株。 </li><li> 30˚Cで48時間、または、単一のコロニーが現れるまで、細胞をインキュベートする。 </li><li>試験管のYEPD液体培地（1％酵母エキス、2％バクトペプトン、2％グルコース）5mlに、単一のコロニーと接種を選んでください。 </li><li>シェーカーやローラードラムのどちらかを使用して一定の撹拌を維持しながら30˚Cでの培養は一晩成長する。 </li><li>一晩培養液50μLと合成完全（SC）培地（表1）5 mLを接種する。一般的に3つのSCの培養物は検査対象の各菌株については寿命の解析の三乗の生物学的な複製を提供するために、各菌株準備されている。 </li><li> 30℃の培養実験全体（一般的に2週間以上）のためのローラードラム上˚Cで一定の攪拌を維持する。 </li></ol>パート2：実行可能性、年齢の点を取る SCメディアの文化の2日後、細胞は静止期にある必要がありますし、最初の年齢の点は取られる準備ができています。それ以降の年齢の点は、最低2週間は、2〜3日ごとに注意が必要です。各年齢の点のための： <ol><li> YEPD 145μLを各ウェルに充填して接種するためのBioscreen 100 -よくハニカムプレートを準備します。のみYEPDとデータ解析のための無細胞以降でうまく満たさ少なくとも一つを残しておくことを忘れないでください。 </li><li>インキュベーターから老化の文化を取り外します。 </li><li>簡潔に渦文化のいずれかをこぼさないように注意しながら、ハニカムプレートに接種される最初の文化。 </li><li>混合培養の5μLを削除し、ハニカムプレートの最初のウェルに、それをピペット。前と5μLアリコートを除去した後、各試験管の口炎。 </li><li>それぞれの文化に対応するウェルの位置をメモしておいているそれぞれの老化の文化について、この手順を繰り返します。同一のよく位置は、実験全体を通して、年齢ポイントごとに、後続のために使用する必要があります。 </li><li>接種が終了するときに30˚Cのインキュベーターに文化を交換してください。 </li></ol>パート3：Bioscreen C MBRのマシンをロード<ol><li>蓋を持ち上げて、インキュベーターのコンパートメントを公開し、サンプルトレイにカバーを取り外します。 </li><li>サンプルトレイ（一つだけプレートを読んでいる場合、左側のスロットを使用）に新たに接種したハニカムプレートを挿入します。 </li><li>サンプルトレイにカバーを元に戻して、インキュベーターのコンパートメントに蓋を下げます。 </li><li>熱伝達流体が最小充填レベルを超えていることを確認してください。低い場合は、付属の熱伝達流体で1000μLピペットを使用して、よりを追加します。 </li><li> Bioscreenソフトウェア&quot;EZExperiment&quot;を使用して、 サッカロマイセスセレビシエに適した成長曲線を得るために次のパラメータを設定します。 <ul><li>サンプル数：メディアまたは200と井戸の数を入力してください</li><li>フィルター：420 - 580nm、広帯域</li><li>温度：30˚C </li><li>実験の長さ：1日、0時間、0秒</li><li>測定間隔：30分</li><li>シェイキング：、連続揺れで、ハイ</li></ul></li><li>朗読を開始する&quot;スタート&quot;をクリックします。 </li></ol>パート4：データ解析一般的に六歳のポイントは、2週間のコースで行われます。年齢の点は日2、4、6、9、11、13で撮影されています。実験的な設計とテストされている菌株に応じて、それは年齢の点、多かれ少なかれ、頻繁にまたは2週間よりも長くかかることが望ましい場合があります。これはデータ分析がより簡単になりますように、各高齢化の文化は、あらゆる年齢の点で同じウェルの位置に対応することをすべての年齢の点などで同じ順序でハニカムプレートをロードすることが重要です。 <ol><li> Bioscreen C MBRのマシンからの出力ファイルを取得します。ソフトウェア&quot;EZExperimentは&quot;Microsoft Excelなどのほか、他のソフトウェアと互換性のあるタブ区切りファイルとしてBioscreenデータを出力します。最初の列のショーは何時にODの測定は、Bioscreenハニカムプレート（図1）で各ウェルを表す以降の列で、実験中に撮影された。 </li><li>すべての列の最初のODの測定値を削除します。この読み方は&quot;ノイズ&quot;です。 </li><li>各列のすべてのOD値から一人でYEPDと同様のOD値を減算することにより、データを正規化する。これは、メディアによるバックグラウンドの吸光度を削除します。 </li><li>伸長曲線をプロットします。曲線は、年齢の関数（図2）としてシフトします。例えば、6つの時間ポイントを介してカラム1（ハニカムプレートの1ウェル）から伸長曲線をプロットすることによって、時間の経過曲線の明確な右シフトがあります。 </li><li>各ウェルに2日目、年齢ポイントの増殖速度に基づいてのために倍の時間を（δ）を計算します。倍加時間iを計算するために使用される式S： <img src="/files/ftp_upload/1156/equat.png" alt="式" /> 外径1とOD 2は連続するOD測定とT 1とT 2の測定値間の時間であることを表す場所。わずか0.2〜0.5のOD値の間の時間を倍に計算する。これらの値の平均は、そのウェルの倍加時間です。わずか0.2〜0.5のOD値の間の時間を倍に計算する。これらの値の平均は、そのウェルの倍加時間です。ほとんどの野生型酵母株は、85〜90分との間の倍加時間の値を与える必要があります。 </li><li>各年齢の点について、最初の年齢ポイント（2日目）からの相対伸長曲線の時間シフト（Δtを）計算する。これを行う簡単な方法は、年齢ポイントの初期と年齢ポイントごとに、その後の間に0.3のODを達成するために各ウェルにかかった時間の長さの違いを決定することです。特定のは、よく0.3のODに達する時間は、OD = 0.3二つの時のポイントブラケットとの間の時間の関数としてLN（OD）に対応する線形回帰式から算出することができる。 </li><li>生存曲線（図3）を生成するために、各年齢の時点で生存率を計算する。 100％の生存率であることが最初の年齢ポイント（2日目）を定義します。年齢ポイントごとに連続するための式を用いて生存率を計算します。 <img src="/files/ftp_upload/1156/equat2.png" alt="第二式" width="245" height="115" /> S Nが生存率である、Δtのnはタイムシフト、およびδ は n倍の時間です。 </li><li>年齢の関数として、生細胞の割合を（またはパーセント）をプロットすることにより生存曲線（図3B）などの各ウェル（またはレプリケート井戸）の所​​望の生成。 </li><li>各ウェルの生存に不可欠（SI）を計算する。 SIは、生存曲線下の面積として定義されており、式で見積もることができます。 <img src="/files/ftp_upload/1156/equat3.png" alt="式（3）" width="400" height="79" /> ここで、年齢nは、年齢の点（例えば2、4、6、9、11、13）であり、S nは、その年齢時点での生存率の値です。 </li><li> SIと生存データから複製井戸の統計的パラメータを決定する。興味のある一般的な統計的なパ​​ラメータは、平均値、中央値、および複製する生物学の各セットのための分散が含まれています。 t検定または類似の分析は、異なる実験群と対照群のSIの一対比較のために使用することができます。それはまた、生物学的に複製平均から​​4.8に記載されて生存曲線を生成することが望ましい場合があります。 </li></ol>パート5：代表的な結果実験終了時には、いくつかの異なる系統や条件の経時的老化の可能性を判断するために十分な生存曲線と実行データの分析をプロットしているでしょう。適切に実行する場合、Bioscreen C MBRのマシンから得られた成長曲線は、図2に示すもののようになっているはずですし、結果の生存曲線は、図3に示すもののようになります。一般的に、ここに記載の条件下で培養した野生型細胞は7日のオーダーの中央値系列の寿命を持つことになります。生存率の大幅な変動が異なる株で、そのような0.05％グルコース培地での成長などのいくつかの条件の下で観察され、生存期間の中央値は30日を超えることができます。 <img src="/files/ftp_upload/1156/1156fig1.png" alt="" /> 図1。Bioscreenプログラム&quot;EZExperiment&quot;からのデータ出力。カラム吸光度の読み取りが行われた時刻が表示されます。連続した列には、老化の培養から採取した細胞を接種したハニカムプレートのウェルを表しています。 <img src="/files/ftp_upload/1156/1156fig2.png" alt="" /> 図2実験の過程で、単一の生物学的複製から伸長曲線。老化培養中の細胞が生存能力を失うように時間をかけての曲線に明瞭な変化があります。最初の時点（2日）と連続する時点間の時間の長さは、その特定の年齢で生存性を決定します。 <img src="/files/ftp_upload/1156/1156fig3a.png" alt="" /><img src="/files/ftp_upload/1156/1156fig3b.png" alt="" /> 図3。 A）生存曲線は、図1から伸長データを使用して生成。 2日目時点を100％の生存率のポイントとして設定されています。B）2つの株の最終的な生存曲線は、同じ実験でテストした。これらの生存曲線は、3つの生物の平均生存率は、それぞれの菌株に対して複製表しています。エラーバーは、生物が複製内の標準偏差を表しています。生存曲線の下の斜線の領域は、歪1の生存に不可欠（SI）を表します。 <table border="1"><tbody><tr><td colspan="2">表1。経時的老化研究のために使用される合成定義ミディアム（株backgrouND BY4743） </td></tr><tr><td>コンポーネント</td><td>濃度（g / L） </td></tr><tr><td> D -グルコース</td><td> 20 </td></tr><tr><td>酵母窒素塩基（-AA/AS） </td><td> 1.7 </td></tr><tr><td> （NH 4）2 SO 4 </td><td> 5.0 </td></tr><tr><td>アデニン</td><td> 0.04 </td></tr><tr><td> L -アルギニン</td><td> 0.02 </td></tr><tr><td> L -アスパラギン酸</td><td> 0.1 </td></tr><tr><td> L -グルタミン酸</td><td> 0.1 </td></tr><tr><td> L -ヒスチジン</td><td> 0.1 </td></tr><tr><td> L -ロイシン</td><td> 0.3 </td></tr><tr><td> L -リジン</td><td> 0.03 </td></tr><tr><td> L -メチオニン</td><td> 0.02 </td></tr><tr><td> L -フェニルアラニン</td><td> 0.05 </td></tr><tr><td> L -セリン</td><td> 0.375 </td></tr><tr><td> L -スレオニン</td><td> 0.2 </td></tr><tr><td> L -トリプトファン</td><td> 0.04 </td></tr><tr><td> L -チロシン</td><td> 0.03 </td></tr><tr><td> L -バリン</td><td> 0.15 </td></tr><tr><td>ウラシル</td><td> 0.1 </td></tr></tbody></table> 注：このレシピは、二倍体BY4743株にauxotrophiesを占める。アミノ酸auxotrophiesは、合成完全培地に5倍の最終濃度を追加することにより補償されるべきである。

<ol>
	<li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+in+yeast:+the+means+to+the+end.">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29 (2008).</li><li>Kaeberlein, M., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of models for human aging. , 109 (2006).</li><li>Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronological+life+span+of+Saccharomyces+cerevisiae.">The chronological life span of Saccharomyces cerevisiae.</a> Aging Cell. 2 (2), 73 (2003).</li><li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span.">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63 (2), 113 (2008).</li><li>Piper, P. W., Harris, N. L., MacLean, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadaptation+to+efficient+respiratory+maintenance+is+essential+both+for+maximal+longevity+and+the+retention+of+replicative+potential+in+chronologically+ageing+yeast.">Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast.</a> Mech Ageing Dev. 127 (9), 733 (2006).</li><li>Fabrizio, P., 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=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166 (7), 1055 (2004).</li></ol>

ここで説明する高スループットの時系列の寿命測定法は高精度と精度を有する株の多数の老化の可能性を定量化するための効果的な方法です。コロニー形成単位をカウントすることで生存を決定するための古典的な手法に比べてこの方法の主な進歩は、（例えば3を参照）、高解像度の成長曲線でを得るためにそのようなBioscreen C MBRのマシンのようなシェーカー/インキュベーター/プ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bacto Peptone</td>
<td>Reagent</td>
<td>BD</td>
<td>211677</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Yeast Extract</td>
<td>Reagent</td>
<td>BD</td>
<td>288620</td>
<td>&nbsp;</td>
<tr>
<td>Difco Agar</td>
<td>Reagent</td>
<td>BD</td>
<td>214530</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Nitrogen Base w/o A.A. and A.S.</td>
<td>Reagent</td>
<td>MidSci</td>
<td>J630-500G</td>
<td>&nbsp;</td>
<tr>
<td>Amino Acids</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Sulfate</td>
<td>Reagent</td>
<td>Spectrum</td>
<td>AM185</td>
<td>&nbsp;</td>
<tr>
<td>Dextrose</td>
<td>Reagent</td>
<td>Fisher</td>
<td>D16-10</td>
<td>&nbsp;</td>
<tr>
<td>Bioscreen C MBR machine</td>
<td>Tool</td>
<td>Growth Curves USA</td>
<td>5101370</td>
<td>&nbsp;</td>
<tr>
<td>Bioscreen 100-well Honeycomb plate</td>
<td>Tool</td>
<td>Growth Curves USA</td>
<td>9502550</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantifying yeast chronological life span by outgrowth of aged cells

出芽酵母<em&gt;サッカロマイセスセレビシエ</em&gt;老化研究の分野において重要なモデル生物であることが実証されています<sup&gt; 1</sup&gt;。複製と時系列のライフスパンは、酵母の老化研究に使用される確立された2つのパラダイムです。経時的老化は時間の細胞の長さによって定義される非分裂、静止のような状態で生き残ることができる、複製老化は、単一の酵母の母細胞が老化する前に生成する娘細胞の数として定義されています<sup&gt; 2</sup&gt;。我々は、時系列寿命の定量的な測定のためのハイスループット法を開発した。この方法は、攪拌下で、一定の温度で定義された培地で細胞を老化伴います。各年齢時点で、細胞の亜集団は、老化培養液から除去され、豊富な増殖培地に接種。高解像度の成長曲線は、Bioscreen C MBRのマシンを使って高齢者の細胞のこの亜集団について得られる。アルゴリズムは、各年齢ポイントでの成長カイネティクスに基づいて、各サブ母集団における生存細胞の相対的な比率を決定するために適用されます。このメソッドは、再現性と精度を維持しながら、他の時系列の寿命のアッセイに比べて大幅に少ない時間とリソースが必要です。このアッセイのハイスループットな性質は、より複雑な生物でさらにテストするために新たな長寿の修飾子を識別するために大規模な遺伝学的および化学的スクリーンを許可する必要があります。

Watch this Scientific Journal Video about Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells at JoVE.com

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

酵母の老化時系列は定常期での時間に関連付けられている細胞の生存率の損失を指します。ここでは、定量的に酵母系列寿命を決定するためのハイスループット法を説明します。

高齢者の細胞の伸長によって酵母年表寿命の定量化

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

quantifying-yeast-chronological-life-span-by-outgrowth-of-aged-cells

Research

JoVE Journal

Biology

16.5K ビュー.  University of Washington. 酵母の老化時系列は定常期での時間に関連付けられている細胞の生存率の損失を指します。ここでは、定量的に酵母系列寿命を決定するためのハイスループット法を説明します。

ビデオ: Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

ライブ固定化酵母細胞の可視化のための高速手法

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

生物学

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

計測線虫（Caenorhabditis elegans）</em固体培地上>寿命

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

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 ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

計測線虫（Caenorhabditis elegans）</em96穴マイクロタイタープレートの>寿命

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

シングル酵母細胞中のMAPK誘導発現の時間的な定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

ヒト末梢血からの血液伸長内皮細胞の生成と文化

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.