この作業は、老年学の賞とNIHグラントMKGSへ1R01AG031108 - 01 NIH訓練助成金P30AG013280でサポートされているのグレン/遠くブレークスルー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：準備線虫の増殖培地（NGM）プレートこのセクションでは、寿命の実験のための固体NGMプレートの準備について説明します。アンピシリン（アンプ）とNGMに追加fluorodeoxyuridine（FUDR）の両方を持っていない添加物、およびアンプ/ FUDRプレートが含まれていては標準のNGMプレート、：基本的な寿命の実験では、プレートの2つのタイプが必要です。アンピシリンは、外国の細菌汚染を防ぐために使用されます。 FUDRは、細胞分裂を阻害卵の生産が減少し、孵化から卵を防ぎます。長寿の分析のためのFUDRの使用は、成人の寿命に影響を与えると成長する幼虫からそれらを分離するために数日ごとにワームを転送する必要がなくなりますしていません。プレートの両方のタイプがEでシードされていますその後、紫外線への曝露によって殺されている大腸菌 OP50細菌、。 <ol><li> NGM（レシピとストレージノート用のセクション5を参照）とラベルのペトリ皿を準備します。あなたは、NGMの10 mLの当たり1 60ミリメートルのペトリ皿が必要に​​なります。以前に調製した固体NGMで開始している場合は、1.2に進みます。あなたが新鮮なオートクレーブNGMで開始している場合は、1.4に進みます。 </li><li>完全に30秒のパルスに高い上に電子レンジで固体NGMを溶かす。構築から加圧ガスの気泡を防ぐために、パルス間の渦巻のメディア。 </li><li> 55 ° Cの水浴中または冷却するためにベンチの上に置いて液体NGM。 </li><li>一度NGMは55に達する℃〜150 mMのFUDRの33μLを追加し、100mLのNGM（アンプ/ FUDRプレート用）と渦巻きは100 mg / mLのアンピシリンを100μLミックスする。追加の薬剤を使わずにNGMプレートは1.5に直接進む。 </li><li>各60ミリメートルのペトリ皿に無菌操作、ピペット10mLのNGMを使用。可能であれば気泡を形成することは避けてください。泡はフォームをすれば、それらはピペットチップを使用してポップすることができます。 </li><li> NGMが固化し、乾燥させるためにふたでベンチでプレートを残す。可能であれば、細菌を追加する前に2日間ベンチでプレートを残すが、プレートがより早く必要とされる場合は、1日に動作します。 </li><li>当日のプレートはE.の新鮮な縞から単一コロニーを、乾燥、種子の液体LB培地を終了する前にLB寒天培地上で大腸菌 OP50細菌を。あなたは、60 mmのプレートあたり一晩OP50文化の少なくとも1 mLを準備する必要があります。 </li><li>場所OP50 37文化℃のシェーカーと細菌は（文化が飽和状態に達するはずです）一晩増殖することができます。 </li><li> 10分3,500 gで回転させてペレットOP50細菌。 </li><li>文化が10倍集中して上清を再懸濁します細菌の90％を削除します。 </li><li>固化NGMプレートの中央に10倍濃縮OP50文化の100μL添加します。優しくスワールプレート、必要に応じて細菌の文化を広げるために（理想的に細菌が板壁の近くに来ることなく、NGMの中心的なエリアをカバーしてください）​​。メディアの表面に欠陥がNGMに巣穴にワームを可能として、NGMの表面にピペットチップを触れないようにしてみてください。 </li><li>細菌の培養は固体NGM（通常は約24時間かかります）で乾燥させるため一晩ベンチ上でプレートを残す。 </li><li>一度乾燥、細菌の増殖を阻止するのに十分な紫外線量にプレートの表面を露出。あなたは、ストラタジーン社製のUV Stratalinker 2400を使用している場合： <ul><li> Stratalinkerにプレートを置き、蓋を取り除く</li><li>ドアを閉めて、Stratalinkerをオンに</li><li>プレス&quot;エネルギー&quot; </li><li> &#39;9999&#39;はキーパッドを使用して入力してください</li><li>プレス&quot;スタート&quot; </li><li>プレートは、約5分間紫外線にさらされる</li></ul></li><li> UV -死菌したプレートは、° Cまでの1ヶ月間4℃逆さまに保存することができます。 </li></ol>パート2：動物の年齢、同期の人口を獲得するために敷設タイミン​​グ卵を実行するこのセクションでは、一般的なハッチ、日付とワームの人口を生成する。これは、生殖アクティブな大人が定義された期間についてはプレート上に卵をできるようにすると、それらの卵を開発することによって実現されます。 <ol><li> （オプション）FUDRせず、新鮮なNGMプレートに約20の若い成虫を転送します。ワームは、細菌性のすべてを通って伝播し、食べることを可能にするために25℃でプレートにしておきます。細菌の食品が消費された後に孵化したワームは、dauer段階成長停止が入力されます。あなたが最初にプレートに転送する方法多くのワームに応じて、週間以内に周りdauer幼虫を見て開始されます。 Dauerの幼虫は、最大1ヶ月間次のステップに使用することができます。 </li><li> FUDRのない新鮮なNGMプレート（任意）転送20〜30 dauer幼虫。食品dauer幼虫の存在下で2日以内に生殖アクティブな大人になると25℃で、その後数日間、生殖アクティブのまま℃に</li><li> FUDRのない新鮮なNGMプレートに10〜15生殖アクティブな大人を転送します。このプレートは、時限の産卵（TEL）プレートと呼ばれます。 </li><li> ℃で6時間tを可能にするために25℃TELプレートを残す彼はワーム産卵のために時間を。 </li><li> TELプレートから成虫を削除します。プレートは、視覚的に大人を削除する前に、卵の検査を行うことができます。ワームは、卵の十分な数を配置していない場合はTELプレートは、成虫を削除する前に24時間の合計に、最大で25 ° Cで残すことができます。 </li><li> 20℃の場所TELプレート° Cの卵が孵化しているとワームはL4幼虫期（これは通常、野生型線虫C. elegansのために2日かかりますが、遅い開発の表現型を有する株のために長い時間がかかることができる）するために開発されるまで。 </li></ol>パート3：寿命のためのスコアの動物彼らは死ぬまでこのセクションでは、パート2からのワームの年齢同期人口に従ってください。ワームは、卵の生産と細菌汚染を防ぐためにアンプ/ FUDRプレート上で維持され、それらが外部刺激に応答しなかったときに死んだと見なされます。 <ol><li>シードアンプ/ FUDRプレートにL4幼虫を移す。テスト対象の各菌株または条件の場合は、それがプレート25〜30ワームを2〜3プレートを設定するのが一般的です。 C.の画像虫のライフステージは、基準（図1）のために用意されています1。 </li><li> 20位のアンプ/ FUDRプレート℃で24時間。 </li><li> 24時間後、視覚的にワーム、メディア、および細菌を評価する。次のいずれかが観察されている場合、新鮮なアンプ/ FUDRプレートにワームを転送します。 <ul><li>ワームは、細菌性のほとんどを食べている。早期寿命の実験のワームの1〜2回の枯渇から細菌を防ぐために週を転送する必要があります。 </li><li>幼虫のかなりの数が存在する。これは通常、ワームは、若年成人の代わりにL4sとしてアンプ/ FUDRプレートに移され、FUDRが発効する前にいくつかの卵を産むことができたていたことを示しています。 FUDRは、一般的に完全な大人に成長してから幼虫を防止しますが、時折少しは大人に成長すると実験動物と混同になります。 </li><li>ライブ/増殖する細菌が観察されています。一般的に、アンプとUV -殺害の組み合わせは生菌は、実験を汚染しないことが保証されますが、時折それが発生します。 </li><li>真菌の増殖は、メディア上に観察される。早期に十分なキャッチした場合、真菌の増殖は通常、ピペットチップやスパチュラを用いて切り取ることができます。一度それは新しいプレートにワームを転送する方が簡単です、直径数ミリメートルより大きくなるように成長する。 </li><li>解剖スコープを使用して、それぞれのワームが生きているのか死んでいるかどうかを決定する。 </li><li>静かにプレートをタップします。それがタップに反応して移動する場合、ワームは生きている。 </li><li>ワームは、頭部領域でのプレート、ズームをタップに応答していない場合。 </li><li>ゆっくりとプラチナの転送ピックとワームの頭をタップします。それが頭を動かすことによって応答しない場合、死んだとしてワームのスコアを獲得。 </li><li>デッドワームは、プレートから除去することができます。 </li></ul></li><li>生きていると死んでいる日付とワームの数を記録。 </li><li> 20℃にプレートを返す</li><li>すべてのワームが死亡するまで繰り返しては、3.3から3.6まで2〜3日おきに繰り返します。 </li></ol>パート4：代表的な結果。 線虫の寿命実験によって生成された生データは、試験した各菌株のための生きていると死んでいるワームの対応する数値と日付のリストです。それぞれの日に死ぬワームの数は通常、生存曲線（図2Bのようにグラフィカルにプロットされている毎日で生きているワームの割合（図2A）を、計算するために反転され、時限産卵の日を0日とみなされます）。研究では個々のワームの寿命は、それぞれの日に死ぬワームの数から計算し、系統間の比較のために平均値と中央値の寿命を計算するために使用することができます。それぞれの日に生きているワームの数のカウントは、寿命の解析に直接使用されません。固体培地上で維持ワームは、時折&quot;逃げる&quot;、またはメディアの下にプレート上下の壁の上のどちらかクロールされます。それぞれの日に生きているワームの数は、どのように多くのワームは、実験の過程を通して逃げたかを判断するために使用することができます。逃げるワームは、通常は分析から削除されます。 N2、Cのベンチマークとして、典型的な中央値の寿命20℃でUV -死菌で維持虫野生型株、° Cは、卵から測定された約25日です。 パート5：ソリューション<table border="0"><tbody><tr><td colspan="2"> 線虫の増殖培地（NGM）、100mLの： </td></tr><tr><td colspan="2">組み合わせる： </td></tr><tr><td> 0.3グラム</td><td> NaClの</td></tr><tr><td> 0.25グラム</td><td>ペプトン</td></tr><tr><td> 2グラム</td><td>寒天</td></tr><tr><td colspan="2"> 40分間オートクレーブし、℃で、[追加]を55に冷ます。 </td></tr><tr><td> 100μL </td><td> 1 M MgSO 4を </td></tr><tr><td> 100μL </td><td> 5 mg / mLのコレステロール</td></tr><tr><td> 100μL </td><td> 1 M塩化カルシウム2 </td></tr><tr><td> 1.625 mLの</td><td> 1.5 M KPI液pH6.0 </td></tr><tr><td colspan="2">液体NGMは、室温でプレートを注いですぐに使用したり、固化させ、長期的に保存することができます</td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> ルリアブロス（LB）、1L： </td></tr><tr><td> 10グラム</td><td>リプトン</td></tr><tr><td> 5グラム</td><td>酵母エキス</td></tr><tr><td> 10グラム</td><td> NaClの</td></tr><tr><td> 10mLの</td><td> 1 MトリスpH8.0 </td></tr><tr><td> 1 L </td><td>脱イオン水</td></tr><tr><td colspan="2">オートクレーブ、室温で保管。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M MgSO 4を 、300mLの： </td></tr><tr><td> 73.95グラム</td><td> MgSO 4を </td></tr><tr><td> 300 mLの</td><td>脱イオン水</td></tr><tr><td colspan="2">オートクレーブ、室温で保管。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 5 mg / mLのコレステロール、200mLの： </td></tr><tr><td> 1グラム</td><td>コレステロール</td></tr><tr><td> 200 mLの</td><td> 100％エタノール</td></tr><tr><td colspan="2">滅菌濾過し、室温で保管してください。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M CaCl 2を 、500mLの： </td></tr><tr><td> 27.75グラム</td><td> CaCl 2の </td></tr><tr><td> 500 mLの</td><td>脱イオン水</td></tr><tr><td colspan="2">滅菌濾過し、室温で保管してください。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1.5 M KPI液pH6.0、1L： </td></tr><tr><td colspan="2">組み合わせる： </td></tr><tr><td> 31.4グラム</td><td> KPI二塩基</td></tr><tr><td> 179.6グラム</td><td> KPI一塩基</td></tr><tr><td> 850 mLの</td><td>脱イオン水</td></tr><tr><td colspan="2">熱KPIは、溶液中に溶解できるように混合しながら。 10 N NaOHでpHを6.0に調整する。 </td></tr><tr><td colspan="2"> 1リットルに脱イオン水を追加します。 </td></tr><tr><td colspan="2">オートクレーブ、室温で保管。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 Mトリス、pH 8.0： </td></tr><tr><td> 60.57グラム</td><td>トリス</td></tr><tr><td> 400mLの</td><td>脱イオン水</td></tr><tr><td colspan="2"> HClでpHを8.0に調整する。 </td></tr><tr><td colspan="2"> 500mLの脱イオン水を追加。 </td></tr><tr><td colspan="2">滅菌濾過し、室温で保管してください。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 150mMのFluorodeoxyuridine（FUDR）、10 mLの： </td></tr><tr><td> 0.3693グラム</td><td> FUDR </td></tr><tr><td> 10mLの</td><td>滅菌脱イオン水</td></tr><tr><td colspan="2"> -20℃で保存</td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 100 mg / mLのアンピシリン（アンペア）、10 mLの： </td></tr><tr><td> 1グラム</td><td>アンピシリン</td></tr><tr><td> 10mLの</td><td>滅菌脱イオン水</td></tr><tr><td colspan="2"> -20℃で保存</td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 50 mg / mLのカルベニシリン（炭水化物）、10 mLの： </td></tr><tr><td> 500mgの</td><td>カルベニシリン</td></tr><tr><td> 10mLの</td><td>滅菌脱イオン水</td></tr><tr><td colspan="2"> -20℃で保存</td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 Mイソプロピルβ- D -チオガラクトピラノシド（IPTG）、10 mLの： </td></tr><tr><td> 2.38グラム</td><td> IPTG </td></tr><tr><td> 10mLの</td><td>滅菌脱イオン水</td></tr><tr><td colspan="2"> -20℃で保存</td></tr></tbody></table><img src="/files/ftp_upload/1152/Figure%201.png" alt="図1" /> 図1℃の明視野像は、卵を含む虫のライフステージ、4つの幼虫（L1 - L4）、および成人。すべてのパネルには、成人男性を（ウッド（1988）からイメージ）を示している、右下を除いて雌雄同体を示す。 <img src="/files/ftp_upload/1152/Figure%202.png" alt="図2" /> 図2℃から代表的な結果DAF - 2遺伝子の突然変異を含む株に野生型株のN2を比較線虫の寿命実験。ワームは、卵、それぞれの日に観測された死者ワームの数、およびそれぞれの日に生きている残りの元のサンプルの割合（のような死者のワームの1日あたりの数から計算）のころからの日数を含めて、収集したデータを示す（A）表。 （B）の生存率は（）で提供される寿命のデータに対応する曲線。 <imgSRC =&quot;/ files/ftp_upload/1152/Figure 3.JPG&quot;ALT =&quot;図3&quot;/&gt; 図3。若年者と高齢成人のCとの間のリポフスチンの代表比較エレガンス 。明視野イメージは、右上、左およびDAPIチャンネル蛍光画像に表示されています。トップパネルは、4日目のワームや下のパネルが11日、古いワームを（のような卵から測定）売主。

<ol>
	<li>Wood, W. B., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 1-16 (1988).</li><li>Sutphin, G. L., 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=Dietary+restriction+by+bacterial+deprivation+increases+life+span+in+wild-derived+nematodes.">Dietary restriction by bacterial deprivation increases life span in wild-derived nematodes.</a> Exp Gerontol. 43, 130-135 (2008).</li><li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans:+a+role+for+heat-shock+factor+and+bacterial+proliferation.">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li><li>Chen, D., Pan, K. Z., Palter, J. E., Kapahi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+determined+by+developmental+arrest+genes+in+Caenorhabditis+elegans.">Longevity determined by developmental arrest genes in Caenorhabditis elegans.</a> Aging Cell. 6, 525-523 (2007).</li><li>Curran, S. P., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+regulation+by+evolutionarily+conserved+genes+essential+for+viability.">Lifespan regulation by evolutionarily conserved genes essential for viability.</a> PLoS Genet. 3, e56-e56 (2007).</li><li>Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+behavior+and+aging+specified+by+mitochondrial+function+during+development.">Rates of behavior and aging specified by mitochondrial function during development.</a> Science. 298, 2398-2401 (2002).</li><li>Hamilton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans.">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19, 1544-1555 (2005).</li><li>Hansen, M., Hsu, A. L., Dillin, A., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+genes+tied+to+endocrine,+metabolic,+and+dietary+regulation+of+lifespan+from+a+Caenorhabditis+elegans+genomic+RNAi+screen.">New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen.</a> PLoS Genet. 1, 119-128 (2005).</li><li>Lee, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+identifies+a+critical+role+for+mitochondria+in+C.+elegans+longevity.">A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.</a> Nat Genet. 33, 40-48 (2003).</li><li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+calorie-independent+life+span+extension+from+dietary+restriction+by+bacterial+deprivation+in+Caenorhabditis+elegans.">Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.</a> BMC Dev Biol. 8, 49-49 (2008).</li><li>Hodgkin, J., Horvitz, H. R., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+Mutants+of+the+Nematode+CAENORHABDITIS+ELEGANS.">Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.</a> Genetics. 91, 67-94 (1979).</li><li>Emmons, S. W., Sternberg, P. W., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. ELEGANS II. , (1997).</li><li>Lipton, J., Kleemann, G., Ghosh, R., Lints, R., Emmons, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+searching+in+Caenorhabditis+elegans:+a+genetic+model+for+sex+drive+in+a+simple+invertebrate.">Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate.</a> J Neurosci. 24, 7427-7434 (2004).</li><li>Davis, B. O., Anderson, G. L., Dusenbery, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans.">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</li><li>Klass, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech Ageing Dev. 6, 413-429 (1977).</li><li>Steinkraus, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+suppresses+proteotoxicity+and+enhances+longevity+by+an+hsf-1-dependent+mechanism+in+Caenorhabditis+elegans.">Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans.</a> Aging Cell. 7, 394-404 (2008).</li></ol>

長寿の遺伝的制御は 、Cで広く研究されている寿命を決定することができる容易さと速さが主な原因虫 、。この記事に記載されているプロトコルは、Cの再現性の寿命のデータを取得するための基本的な枠組みを説明しエレガンスとはrelatednematodeの種にも適用できます。2は 、これらの手順は、さまざまな条件下での寿命を測定するために適合させることが?...

<!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>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0145-17-0 (214530)</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
<tr>
<td>BactoTryptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0123-17-3 (211705)</td>
<td>&nbsp;</td>
<tr>
<td>BactoPeptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0118-17-0 (211677)</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9699248 (1332-01)</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>C0109</td>
<td>&nbsp;</td>
<tr>
<td>Cholesterol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C75209</td>
<td>&nbsp;</td>
<tr>
<td>Fluorodeoxyuridine (FUDR)</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl &#x03B2;-d-Thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>I2481C5</td>
<td>&nbsp;</td>
<tr>
<td>KPi Dibasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087862 (3252-01)</td>
<td>&nbsp;</td>
<tr>
<td>KPi Monobasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087861 (3246-01)</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9561800 (2500-01)</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S251</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0886-17-0 (288620)</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring caenorhabditis elegans life span on solid media

老化は細胞成分と死亡率をもたらす細胞内小器官の進行性の悪化によって特徴付け変性過程です。線虫<em&gt;線虫（Caenorhabditis elegans）</em&gt;老化の生物学を研究するために使用される主なモデルとして登場しました。事実上すべての生物学的なサブシステムは、加齢に伴って機能低下を起こしているので老化の合計率を考慮した場合、寿命は、関心の主要エンドポイントです。線虫では、寿命は一般的に動物が外部刺激に応答残る日数として定義されます。線虫は、液体培地で、プレートの固体培地上でいずれかを伝達することができます、そしてテクニックは、両方の条件下での寿命を測定するために開発されている。ここでは、固体の線虫の増殖培地上で維持線虫の寿命を測定するための一般的なプロトコルを提示し、UV -死菌の飼料を与えた。これらの手順は、簡単に、生菌、食事制限、およびRNA干渉で構成される食事療法を含め、様々な一般的な条件下でアッセイの寿命に適合させることができます。

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span on Solid Media at JoVE.com

Measuring Caenorhabditis elegans Life Span on Solid Media

この記事では、UV殺菌細菌食品を固体培地上で維持線虫の寿命を測定するための一般的なプロトコルを提示する。

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-caenorhabditis-elegans-life-span-on-solid-media

Research

JoVE Journal

Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

29.7K ビュー.  University of Washington. この記事では、UV殺菌細菌食品を固体培地上で維持線虫の寿命を測定するための一般的なプロトコルを提示する。

ビデオ: Measuring Caenorhabditis elegans Life Span on Solid Media

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

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 ...

生物学

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 ...

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

固体プレートベースの食餌制限線虫（Caenorhabditis elegans）</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培養<em&gt;線虫（Caenorhabditis elegans）</em &gt;微小粒子衝撃による無菌液体培地およびトランスジェニックワームの作成に

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

の酸化ストレス抵抗を測定<em&gt;線虫（Caenorhabditis elegans）</em&gt; 96ウェルのマイクロタイタープレート中

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

カエノハブディティス・エレガンスを使用して組織特異的シャペロン相互作用をスクリーニングする

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

刺激されたラマン散乱顕微鏡を用いた カエノラム型エレガンス における脂質蓄積ダイナミクスの無標識イメージング

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegansにおける凝集定量の自動化

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

計測線虫（Caenorhabditis elegans）寿命