この作品は、NIHとACS、T32CA009135含む、CA133027、CA070723、およびCA022443からの補助金によって賄われていた。ビルサグデンは、アメリカ癌協会の研究教授である。

1。可視プラスミドを有する細胞のエンジニアリング<ol><li>視覚化するプラスミドにラクトースオペレーター配列（LACO）の約250部を含むDNA断片を導入するために標準の制限酵素消化または相同組換え技術を使用しています。私たちのプラスミドは約170 kbpのバクミドだっおよびハイグロ12にカポジ肉腫関連ヘルペスウイルスの全ゲノム（KSHV）とエンコードされた抵抗を含んでいた。 </li><li>リポフェクタミン2000を用いたトランスフェクションにより、哺乳動物細胞へLACO含有プラスミドDNAを導入。我々は、25μlのリポフェクタミン2000、0.5ミリリットルのOptiMEM、および3.5ミリリットルDMEMで精製プラスミドDNA10μgを4時間の場合は60 mmディッシュでHeLaおよびSLK細胞をトランスフェクションした。 </li><li> 10％ウシ胎児血清および48時間インキュベート細胞と5ミリリットルDMEMに培地を変更します。 </li><li>プレート導入ナンプラーための抗生物質の選択を含む培地中で大きな皿や培養細胞における低密度での細胞SMID、我々は3週間300μg/ mlのハイグロマイシンを10％ウシ胎児血清を含むDMEM中で細胞を選択した。 </li><li>新しい培養皿にハイグロマイシン耐性コロニーを転送するために、滅菌したトリプシンに浸したろ紙の小片を使用しています。 </li><li>トランスフェクトされたクローンは皿を埋めるために成長しているときは、プラスミドLACOのポリマーが均一に、予想されたサイズのあることを確認するブロッティング、制限消化し、サザンのためにDNAを単離する。 </li><li>プラスミドはlacIの融合を発現するように設計されていない場合は、そのようなLacIリ-tdTomatoとして赤色蛍光タンパク質と融合ラクトースリプレッサー（LACI）をコードするレトロウイルスを適切なクローンを感染させる。我々はtdTomatoはなく長い時間13を介して複数のエクスポージャーから生じるであろうphotoxicityを最小限にするために緑色に近い蛍光を発するタンパク質を使用しています。 </li><li>バインディングからの融合タンパク質をブロックするように200μg/ mlのイソプロピル-BD-ガラクトシド（IPTG）の存在下でLacIリ融合タンパク質が導入されると、細胞培養DNA。 </li><li>文化少なくとも3日間の細胞は、lacI-tdTomatoタンパク質の発現を可能にする。 </li><li>バインドされていない融合タンパク質の最小限のバックグラウンドレベルとの明確なシグナルを明らかLacIリ-tdTomatoの最適レベルとのそれらのためのスクリーンのクローン（IPTGを洗い流し、下記のようにセルを表示することによって）。 </li></ol> 2。イメージングシステムの開発当社のイメージング·システムは、電動ステージと計画Aochromat 63x/1.4油浸対物装備ZEISS AXIOVERT 200Mで構成されています。蛍光励起光がコリブリシステムのLEDが &quot;昼白色&quot;によって提供されています。 1024 EMCCDカメラ：発光がCascadeIIによって検出されます。これは、信号を増幅するゲイン·レジスタを用いて冷却、裏面入射型カメラです。暗電流は、毎秒画素あたり0.061電子であり、量子効率は0.90より大きい。システムはSmartExperimentsモジュールを含む、AxioVisionの4.8ソフトウェアによって制御されます。 tdTomaの検出のための我々はそのために通過した光の85％が蛍光と放出された光の95％を励起することができるツァイスフィルターセット75HEを使用すると、発光フィルターを通過します。 <ol><li>複数の長い時間間隔で連続して画像の自動取得のために敏感な信号の検出、特定の励起と高速シャッター、電動ステージとフォーカスアーム用LED光源、およびソフトウェア制御用倒立顕微鏡とEMCCDカメラとイメージングシステムを組み立てるZ-スタック。システムは、振動から隔離される空気テーブルの上に座ってください。 </li><li> 37℃、加湿チャンバー内の5〜10％のCO 2で細胞を維持するために、ステージトップインキュベーターを組み立てる。実験中に動きのアーチファクトを最小限に抑えるために安定した温度に来るシステムのための十分な時間を確保します。 </li><li>サンプルから熱を吸い上げるから客観を防ぐために客観的なため、加熱されたカラーを使用しています。 </li></ol> 3。標識されたDNAの可視化<ol><li> 35ミリメートルの板細胞セルのペアが重ならずに8月16日細胞のコロニーに分割することができます10％未満の合流点の密度でガラスボトムディッシュ、。生存細胞が分裂する時間を持つようにイメージを作成する前に、この少なくとも24〜48時間を行う。 </li><li>撮影開始前に1〜3時間、選択的薬剤およびIPTG両方を欠いている培地でプレートを3回すすいでください。この手順では、離れて、非生存細胞を洗浄およびlacI融合タンパク質はプラスミド中LACOサイトをバインドすることができます。 </li><li> 10％ウシ胎児血清および25mM HEPESで2ミリリットルDMEMで培養液を交換してください。 </li><li> 35mmディッシュ用取付リングに延伸CultFoilと培養皿の上部を元に戻します。このカバーは、時間をかけて塩濃度を増加させ、細胞を殺す培地の蒸発を抑制します。 </li><li>セルを表示し、ビューの複数のフィールド内のセルの上部と下部の焦点面を決定する微分干渉コントラスト（DIC）のイメージングを使用しています。各フィールドで、セルの上部から焦点面に道の約1/3を下に移動します。 x、yを保存し、zが保存されてステージ位置のリストに座標。 注：プラスミド信号は細胞があるlacI融合タンパク質を適切な波長のLEDによって照らされる接眼レンズを介して表示されない場合があります。弱い信号だけ時間をかけて信号の統合を可能にし、したがって効果的に弱い信号を増幅EMCCDカメラの使用、と見えるかもしれません。 </li><li>顕微鏡制御ソフトウェアでは、保存された各ステージの位置で実行される画像のzスタックを定義します。 z次元14に近似ナイキスト·サンプリングにあなたをできるようになります0.35から0.50μmのAZのステップサイズを選択してください。細胞周期の間の細胞の動きに従い、これらのスライスは、検出を可能にし、また画像スタックの取得後の処理（デコンボリューション）で使用され、細胞が置かれている面の上下に &quot;余分な&quot;スライスが含まれています。この包含は意志セルの厚さに応じて、40から60までの画像をスタックにつながる。 </li><li>顕微鏡制御ソフトウェアでは、（プラスミドを可視化するための10から60分間隔で30分間隔（長時間にわたって細胞分裂および移行を追跡するため）と蛍光画像でZ-スタックの明視野像を収集するために、時系列の実験を定義細胞内で）。それは長い間実験の過程で光毒性を不必要に細胞を公開することができます標準の明視野像、より多くの光を必要とするため、実験中にDICのイメージングを使用しないでください。 </li><li>ソフトウェア制御の実験を開始します。システムが妨害されないことを確認します（等、過剰な室内光にさらされ、バンプ）実験中。 </li><li>必要に応じて、実験中に加湿システムで水を補給してください。 </li></ol> 4。画像の取得後の処理<ol><li>各zスタックと時間ポイントの画像を確認してください。の不要な領域を切り抜く次のステップでコンピュータの処理時間を短縮するための画像。 </li><li>各zスタック15で原点の画素に信号強度を再割り当てするにはデコンボリューションアルゴリズムを使用します。このデコンボリューションは、イメージングシステムのための理論的な点広がり関数に基づくこ​​とができますが、それは、イメージング蛍光ミクロスフェアによる特定のシステムの点広がり関数を測定し、デコンボリューションアルゴリズムでは、この測定値を使用することが好ましい。私たちは、イメージングシステムの点広がり関数を測定し、適用AxioVisionのソフトウェア（AxioVisionのソフトウェアの説明、ツァイス）の最尤反復アルゴリズムを拘束されています。 </li><li>細胞周期と娘細胞へのプラスミドのパーティションの分割中にプラスミドの強度変化と動きを追跡するために画像を調べます。 </li></ol>

<ol>
	<li>Cesarman, E., Chang, Y., Moore, P. S., Said, J. W., Knowles, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-associated+herpesvirus-like+DNA+sequences+in+AIDS-related+body-cavity-based+lymphomas.">Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.</a> N. Engl. J. Med. 332, 1186-1191 (1995).</li><li>Chang, Y., 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=Identification+of+herpesvirus-like+DNA+sequences+in+AIDS-associated+Kaposi's+sarcoma.">Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.</a> Science. 266, 1865-1869 (1994).</li><li>Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology 2 volume set. , (2006).</li><li>Adams, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+latent+Epstein-Barr+virus+genomes+in+Raji+cells.">Replication of latent Epstein-Barr virus genomes in Raji cells.</a> J. Virol. 61, 1743-1746 (1987).</li><li>Humme, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EBV+nuclear+antigen+1+(EBNA1)+enhances+B+cell+immortalization+several+thousandfold.">The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 10989-10994 (1073).</li><li>Verma, S. C., Choudhuri, T., Robertson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimal+replicator+element+of+the+Kaposi's+sarcoma-associated+herpesvirus+terminal+repeat+supports+replication+in+a+semiconservative+and+cell-cycle-dependent+manner.">The minimal replicator element of the Kaposi's sarcoma-associated herpesvirus terminal repeat supports replication in a semiconservative and cell-cycle-dependent manner.</a> J. Virol. 81, 3402-3413 (2007).</li><li>Yates, J. L., Guan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus-derived+plasmids+replicate+only+once+per+cell+cycle+and+are+not+amplified+after+entry+into+cells.">Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells.</a> J. Virol. 65, 483-488 (1991).</li><li>Ye, F. C., 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=Disruption+of+Kaposi's+sarcoma-associated+herpesvirus+latent+nuclear+antigen+leads+to+abortive+episome+persistence.">Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence.</a> J. Virol. 78, 11121-11129 (2004).</li><li>Robinett, C. C., 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=In+vivo+localization+of+DNA+sequences+and+visualization+of+large-scale+chromatin+organization+using+lac+operator/repressor+recognition.">In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.</a> J. Cell Biol. 135, 1685-1700 (1996).</li><li>Nanbo, A., Sugden, A., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coupling+of+synthesis+and+partitioning+of+EBV's+plasmid+replicon+is+revealed+in+live+cells.">The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells.</a> Embo. J. 26, 4252-4262 (2007).</li><li>Herndier, B. G., 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=Characterization+of+a+human+Kaposi's+sarcoma+cell+line+that+induces+angiogenic+tumors+in+animals.">Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals.</a> Aids. 8, 575-581 (1994).</li><li>Zhou, F. C., 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=Efficient+infection+by+a+recombinant+Kaposi's+sarcoma-associated+herpesvirus+cloned+in+a+bacterial+artificial+chromosome:+application+for+genetic+analysis.">Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.</a> J. Virol. 76, 6185-6196 (2002).</li><li>Shaner, N. C., 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Pawley, J. B., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Points,+pixels,+and+gray+levels:+digitizing+image+data.">Points, pixels, and gray levels: digitizing image data.</a> Handbook of Biological Confocal Microscopy. , 59-79 (2006).</li><li>Cannell, M. B., McMorland, A., Soeller, C., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+enhancement+by+deconvolution.">Image enhancement by deconvolution.</a> Handbook of biological confocal microscopy. , 488-500 (2006).</li></ol>

特別な利害関係は宣言されません。

ここで説明する方法は、複数の世代にまたがる時間をかけて生きた哺乳動物細胞におけるプラスミドをフォローするために使用できます。励起光への曝露を制限することによって、我々は、少なくとも72時間、3細胞分裂を表す16から32細胞のコロニーを通って新しく分割ペアからセルをフォローするために、これらの技術を使用している。これらの実験は、このような定量的PCR、サザンブロッ...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11965 </td><td></td></tr><tr><td> OptiMEM中</td><td> GIBCO </td><td> 31985 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td> Hyclone社</td><td> SH30910.03 </td><td></td></tr><tr><td>ペニシリン</td><td>シグマ</td><td> P3032 </td><td></td></tr><tr><td>硫酸ストレプトマイシン</td><td>シグマ</td><td> S9137 </td><td></td></tr><tr><td>ハイグロマイシン</td><td>カルビオケム</td><td> 400050 </td><td> SLKのための400μg/ mlの、HeLa細胞では300μg/ mlの</td></tr><tr><td>イソプロピル-BD-ガラクトシド（IPTG） </td><td>ロッシュ</td><td> 10 724 815 001 </td><td>最終濃度が200μg/ mlの</td></tr><tR&gt; <td>リポフェクタミン2000 </td><td>インビトロジェン</td><td> 11668-027 </td><td></td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td></td></tr><tr><td>ガラスボトムディッシュ</td><td>マテック</td><td> P35G-1.5-10-C </td><td></td></tr><tr><td> CultFoil </td><td> Pecon </td><td> 000000-1116-08 </td><td></td></tr><tr><td> AXIOVERT 200M </td><td>ツァイス</td><td></td><td></td></tr><tr><td>コリブリ</td><td>ツァイス</td><td> 423052-9500-000 </td><td></td></tr><tr><td>中性の白色LED </td><td>ツァイス</td><td> 423052-9120-000 </td><td></td></tr><tr><td>フィルターキューブ75 HE </td><td>ツァイス</td><td> 489075-0000-000 </td><td></td></tr><tr><td>プラン·アポクロマート63x/1.4客観</td><td>ツァイス</td><td> 440762-9904-000 </td><td></td></tr><tr><td> CascadeII：1024 EMCCDカメラ</td><td> Photometrics </td><td> B10C892007 </td><td></td></tr><tr><td> Tempcontrolミニ</td><td>ツァイス/ Pecon </td><td> 000000-1116-070 </td><td></td></tr><tr><td> Tempcontrol 37から2デジタル</td><td>ツァイス/ Pecon </td><td> 000000-1052-320 </td><td></td></tr><tr><td> CTIコントローラー3700デジタル</td><td>ツァイス/ Pecon </td><td> 411856-9903 </td><td></td></tr><tr><td>客観ヒーター</td><td>ツァイス/ Pecon </td><td> 440760-0000-000 </td><td></td></tr><tr><td>ステージ加熱インサートP </td><td>ツァイス/ Pecon </td><td> 411861-9901-000 </td><td></td></tr><tr><td>ステージトップインキュベーターS </td><td>ツァイス/ Pecon </td><td> 411860-9902-000 </td><td></td></tr><tr><td>加湿システム</td><td>ツァイス/ Pecon </td><td> 000000-1116-065 </td><td></td></tr></tbody></table>

monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy

いくつかの天然に存在するプラスミドを、哺乳動物細胞内に保持されます。これらのうち、複数のヒト悪性腫瘍1-3引き起こすエプスタイン-バーウイルス（EBV）およびカポジ肉腫関連ヘルペスウイルス（KSHV）を含むガンマヘルペスウイルスのゲノムは、されています。これら二つのゲノムは単一のウイルス蛋白質と細胞複製機構を使用して、それぞれの、ライセンスされた方法で複製され、その欠けている伝統的なセントロメア4月8日にもかかわらず、細胞分裂の際に娘細胞に渡されます。 多くの仕事は、サザンブロッティングやin situハイブリダイゼーション（FISH） 蛍光 inなどのメソッドを使用して、これらのプラスミドゲノムの複製を特徴付けるために行われている。これらの方法は、しかし、制限されています。定量PCRおよびサザンブロットは、細胞集団における細胞あたりのプラスミド数の平均値に関する情報を提供します。 FISHは平均数とプラスミドの分布の両方を明らかに単一セルアッセイである<html>の細胞あたりの細胞集団でなく調べセルの親や子孫の情報を全く許さない、静的なものである。 ここでは、生きた細胞内のプラスミドを可視化するための方法を説明します。このメソッドは、関心9のプラスミド内に複数のサイトへの蛍光標識ラクトースリプレッサータンパク質の結合に基づいています。目的とするDNAをラクトースオペレーター（LACO）配列の約250タンデムリピートを含むように設計されています。 LACOは、具体的に蛍光タンパク質に融合させることができるラクトースリプレッサータンパク質（LACI）によってバインドされています。融合タンパク質は、いずれかの人工プラスミドまたはレトロウイルスベクターにより導入されたから発現させることができる。このように、DNA分子は、蛍光標識、したがって、蛍光顕微鏡を介して、目に見えるようになるされています。プラスミドが閲覧できるようになるまで融合タンパク質は、IPTGの存在下で培養することによりプラスミドDNAを結合からブロックされています。 ontentは &quot;&gt;このシステムは簡単にトランスフェクションし、理想的な細胞が接着されています。それらの合成の特性を明らかにし、娘細胞に分割、プラスミドは数世代を通して生きた細胞で監視することができ、大規模な核を持っているこの技術はあることを決定するために使用されていますEBV由来プラスミドの84％は、HeLa細胞に娘細胞に忠実に、各世代を合成し、新たに合成されたプラスミドパーティションの88％されており、これらのEBVプラスミドのペアがでそれらの合成の後につながまたは姉妹染色分体に関連付けられていることが観察されたS-彼らは後期10で分離された姉妹染色分として分離するのを見たまで。メソッドは現在、HeLa細胞とSLK細胞におけるKSHVゲノムの複製を研究するために使用されています。HeLa細胞は、ヒト上皮細胞を不死化され、SLK細胞が内皮ヒト不死化している段階細胞。SLK細胞はもともとKSHVの病変に由来したものの、ヒーラもSLKのセルライン自然にハーブもORS KSHVゲノム11。ウイルス複製の勉強に加えて、この可視化技術は、合成、ローカリゼーション、および他の組換えプラスミドDNAのパーティション化に様々なDNA配列の要素の追加、削除、または変異の影響を調査するために使用することができます。

<html>代表的な実験で取得したZ-スタックの最大強度の予測を図3に示します。典型的なプラスミド信号は、単一またはプラスミドのクラスタを表しているかどうかに応じてzスタックの3-8スライスに存在しています。細胞が分裂期に達するまでのEBVおよびKSHV由来プラスミドの場合、信号が細胞内でゆっくりと移動します。細胞自体は、実験のコースを上に移行されます。彼らはまた...

Watch this Scientific Journal Video about Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy at JoVE.com

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

生きた細胞内の個々のDNA分子を観察する方法が記載されている。技術は目的のDNAに組み換え結合部位に蛍光標識lacリプレッサータンパク質の結合に基づいています。この方法は、時間をかけて生きている細胞内の多くの組換えDNAをたどるように適応させることができる。

蛍光顕微鏡法による、複数世代にわたってライブ哺乳類細胞におけるプラスミドの複製を監視

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

monitoring-plasmid-replication-live-mammalian-cells-over-multiple

Research

JoVE Journal

Biology

11.7K ビュー.  University of Wisconsin - Madison. 生きた細胞内の個々のDNA分子を観察する方法が記載されている。技術は目的のDNAに組み換え結合部位に蛍光標識lacリプレッサータンパク質の結合に基づいています。この方法は、時間をかけて生きている細胞内の多くの組換えDNAをたどるように適応させることができる。

ビデオ: Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

広視野顕微鏡を用いて長期的なイメージング哺乳類細胞

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

蛍光顕微鏡による可視化単分子のDNAの複製

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

単分子バイオセンサーを用いた生細胞中のシグナル伝達事象をリアルタイムで監視するための顕微鏡のFRET

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

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) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

蛍光標識ナノ粒子を用いたマウス、ヒトおよびブタ起源のライブ細胞の細胞内遺伝子発現の検出

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...