著者らは、スウェーデンの研究評議会、スウェーデン戦略研究財団、スウェーデン癌の社会、スウェーデンの小児がんの社会、オーケWibergs基盤とJeanssons基盤によってサポートされています。

1。細胞にプローブを導入する<ol><li> FRETに基づくプローブとプラスミド耐性を付与するとの同時トランスフェクション細胞。目的の細胞型で効率的なトランスフェクションの方法を選択してください。 U2OS細胞の場合は、標準的なリン酸カルシウムトランスフェクション法では十分な結果が15を与える。 </li><li>少なくとも7日間に適切な抗生物質でセルを選択します。これは豊かにプローブを発現する細胞の量をし、深刻な細胞周期に影響を与える毒性発現レベル、または発現レベルと細胞の量を制限します。 </li><li> （省略可能）安定したクローンを選択してください。 </li><li>固定またはライブセルを使用して、CFPとYFPの両方がほぼ同じ比率ですべての細胞に存在することを確認してください。 </li><li>一部の細胞だけCFPまたはYFPを含む場合には、組換えは可能性が発生しました。組換えは、細菌の増殖の間またはプローブのトランスフェクション後にどちらかが発生することができ、後者の場合にはプラスミドの品質に依存することがあります。プラスミドpを繰り返す償いと問題が解決しない場合はトランスフェクション前にプラスミドを線形化します。 </li></ol> 2。レシオメトリックを監視すると、FRETの<ol><li>チャンバー内にマウントするためのガラスボトムディッシュ、またはカバースリップ上にシード細胞。食器/カバースリップがない1.5（170μm厚）がないことを確認してください。 </li><li> 37℃に設定した温度制御による電動落射蛍光顕微鏡でフェノールレッドを増殖培地で細胞をマウントします。 CO 2によってまたはそのようなリーボウィッツ- 15などのCO 2 -独立系メディアを使用するかのいずれか、培地のpHの調節を確認してください。 </li><li>透過光を用いて細胞に焦点を当てる。蛍光灯を用いて細胞を直視しないでください。 </li><li> YFPの励起（YFPex）とYFPエミッション（YFPem）フィルタと、適切なダイクロイックミラーを使用して12または16ビットの画像を取得することから始めます。それでも、必要な細胞内解像度を可能にする使用可能な最大のビニングを使用してください。露光時間や強度を減少させる高ビニングを使用すると、光毒性を避けるために不可欠です。 </li><li>私asureまたは細胞を欠いているエリアでラフ平均、最小値および最大ピクセル値を測定または推定することによりバックグラウンドの強度とおおよそのノイズを推定する。セル内の平均的な信号はおよそバックグラウンド強度に加え、最大および最小のバックグラウンド強度の間に5〜10倍の差になるように露光時間とNDフィルターを変更します。 </li><li>飽和するために近くに配置されていないことのイメージを確認します。画像の最大強度は、細胞が有糸分裂を入力すると強度の違いを考慮してダイナミックレンジ（12ビットの画像で、例えば最大2000）の半分未満でなければなりません。 </li><li> CFPの励起とYFPの発光フィルターと、適切なダイクロイックミラーを使用して、ステップ2.4​​から2.6を繰り返します。 </li><li> 2.7ポイント2.4で使用される地域を除く、トランスフェクションされた細胞で複数の地域を選択し、その最大強度を確保するためには、すべてのセルのダイナミックレンジの半分以下です。 </li><li>すべての4つの画像を取得タイムラプス実験を開始YFPexを使用して5分​​ - YFPemとCFPex - YFPemフィルタセット。 </li></ol> 3。条件の確認と最適化<ol><li>取得した画像を開いて、有糸分裂から、トランスフェクトプローブを発現する細胞のための有糸分裂への細胞のサイクルタイムを監視します。 </li><li>使用する細胞の種類の公開データに測定された細胞周期の時間を比較。また、フィルムは、参照セルのサイクルタイムを取得する唯一の透過光の1つのイメージ（例えば、DICまたは位相コントラスト）時間ごとに取得することにより細胞をトランスフェクトされていない。 </li><li>有糸分裂から分裂までの時間は、細胞周期のタイミングを参照するために該当する場合においては、ステップ4に進みます。あるいは難しい露出を使用して、再取得の画像、より多くの時間がポイントまたは複数のz -レベルとステップ3を繰り返します。 </li><li>細胞周期のタイミングを参照するには対応していない有糸分裂から分裂までの時間のケースでは、映画は最後にのみ透過光の1つのイメージ（例えば、DICまたは位相コントラスト）は、1つの画像に続いてすべての時間を獲得することで細胞をトランスフェトランスフェクションされた細胞を識別するための実験の。 </li><li>トランスフェクションされた細胞はまだ長く、細胞周期の時間を表示する場合、手順1を繰り返しますが、少ないプローブをトランスフェクトまたはプローブの非常に少量を含む安定版（stable）クローンを選択します。 </li><li>トランスフェクションされた細胞は、蛍光灯のない状態で正常な細胞周期の時間を表示する場合、画像間のビニング、NDフィルター、露光時間、および時間を変更することで、手順2が下側の露出を繰り返す。 </li></ol> 4。 FRETはAnalysing <ol><li>分析は、顕微鏡専用の多くのソフトウェアで実行することができます。ここでは、フリーウェアのImageJ（使用して分析を実行する方法について説明します。 <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ijを</a>プラグイン比プラス（活用） <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plusを。HTML</a> ）。 </li><li> ImageJの中の画像を開きます。ケースではあなたのイメージが多色のSTACの形式になっていますK（例：Deltavision）は、最初のhyperstackに変換することで、個々のチャネルを分離する：hyperstackするImage - hyperstack -スタックして、その後使用して単一のチャネルスタックにそれらを分割： 画像- hyperstack -減らす次元 </li><li>それが唯一の比率は0と1の間の範囲内にないことを保証することで、画像の視覚的な明瞭さを高めるために意図されているため、このステップは、オプションですプロセス-数学-乗算 ：使用して3でYFPemスタック- YFPexを掛けます。 </li><li> YFPexに - YFPemスタック、細胞を欠いているが、それは測定しようとする細胞に近い地域に関心領域（ROI）を描く。バックグラウンドと信号強度の両方が一般的に画像の中心に最も強いのと同じイメージ内の別の細胞は、さまざまな地域が必要となる場合があります。 </li><li> ROIマネージャーにROIを追加： 分析-ツール- ROIマネージャー 。 </li><li> 分析-測定 ： - YFPemとCFPex - YFPemスタックYFPex両方のROIの平均強度を測定</em&gt;。 </li><li>複数のタイムポイントで繰り返すとその目的の細胞に近いバックグラウンド強度がフィルム全体に似ていることを確認します。 </li><li> 分析-セットの測定値- MinとMaxのグレー値 ：最小の強度を含むように測定値を設定します。 </li><li>細胞のほとんどをカバーする関心領域を描くとYFPex両方で最小限の強度を測定する - YFPemとCFPex - YFPemスタック。 4.6から測定された最小の強度とバックグラウンド強度の差を取ると2で割ります。これは、クリッピングの値の開始見積もりを提供します。 </li><li>オープン比プラス。 YFPemとCFPex - - YFPemスタックYFPexを選択します。比、選択CFPex FRETのために - スタック1としてYFPem - スタック1としてYFPemを、逆にリン酸化によって効率が低下するのFRETプローブ、選択YFPexを可視化比率をFRETのために。 </li><li>測定されたバックグラウンド強度とクリッピングの値を挿入します。 </li><li>結果として得られる比スタックのスケーリングを設定し、適切な外観のuを適用する比の変化を視覚化するpは、テーブル（LUT）。 </li></ol> 5。代表的な結果 PLK1活動は、G2期および有糸分裂の間にピークに核内で最初に表示されているです。図3は、セクション2で説明したように最小限の光毒性の設定を使用して実験を示しています。これは代表的な初期の結果であることに注意し、画像間の露光条件や時間が雑音比や時間分解能の信号を増加するように変更することができますしてください。プローブを発現する細胞の大部分は、撮影条件やプローブの発現レベルが細胞周期のタイミングに影響しないことを示す、20〜25時間の細胞周期の時間で増殖することを図3に示す。かなりのノイズがあるが、PLK1活動の傾向G2で増加し、有糸分裂14にピークがはっきりと（図3A）表示されます。ここで、生データを処理するわけで平均的な倒立RAのカイモグラフ（図3B）、または定量化として提示フィルタリング<html>酸化チタン（図3C）は、透明度を高めることができます。 <img alt="図1" src="/files/ftp_upload/3410/3410fig1.jpg" /> 図1 FRETに基づくプローブの原理キナーゼとホスファターゼの活動を監視する。通常2つのフルオロフォア、CFP（青）とYFP（緑色）は、リン結合ドメイン（オレンジ）とリン酸化可能なシーケンス（黄色）で接続されています。これにより、プローブのコンフォメーション変化を誘導するリン結合ドメインに結合するリン酸化（赤）を仲介する、。 CFPとYFP間のFRET効率に影響を与えるtwoフルオロフォア間の距離や方向の違いにおけるコンフォメーション変化の結果。 FRETは、刺激的なCFPとYFPの監視の放出（点線）によって可視化することができる。 <img alt="図2" src="/files/ftp_upload/3410/3410fig2.jpg" /> 図2実験手順の概略図概略。 p_upload/3410/3410fig3.jpg&quot;/&gt; 図3。PLK1の活性は、G2期および有糸分裂の間にピークに核内で最初に表示されているです。 FRETに基づくプローブの監視PLK1活動9,14の発現U2OS細胞は20倍、NA 0.7空気の目標と水銀ランプを装備Deltavisionスペクトリスイメージングシステムを用いて60時間撮影されました。撮像条件は、4倍ビニングとNDフィルターを使用して、2章で概説したように、最小限の光毒性を引き起こすために選ばれたその入射光のブロック99％。 、逆の偽色の表現は、4部門を通じて1つのセルに続く、比のFRET。 B、​​平均値フィルタを適用した後に示すようにセルのキモグラフ。 C、反転の定量化は、A. D、娘細胞の分裂を含む50のFRETプローブを発現する細胞、の累積有糸分裂のエントリに示されている細胞の比率のFRET。

<ol>
	<li>Sun, Y., Wallrabe, H., Seo, S. A., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+microscopy+in+2010:+the+legacy+of+Theodor+Forster+on+the+100th+anniversary+of+his+birth.">FRET microscopy in 2010: the legacy of Theodor Forster on the 100th anniversary of his birth.</a> Chemphyschem. 12, 462-474 (2011).</li><li>Allen, M. D., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+dynamics+of+protein+kinase+A+activity+visualized+by+FRET-based+reporters.">Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.</a> Biochem. Biophys. Res. Commun. 348, 716-721 (2006).</li><li>Kunkel, M. T., Ni, Q., Tsien, R. Y., Zhang, J., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+dynamics+of+protein+kinase+B/Akt+signaling+revealed+by+a+genetically+encoded+fluorescent+reporter.">Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter.</a> J. Biol. Chem. 280, (2005).</li><li>Violin, J. D., Zhang, J., Tsien, R. Y., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+reporter+reveals+oscillatory+phosphorylation+by+protein+kinase.">A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase.</a> C. J. Cell. Biol. 161, 899-909 (2003).</li><li>Kunkel, M. T., Toker, A., Tsien, R. Y., Newton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+regulation+of+protein+kinase+D+revealed+by+a+genetically+encoded+kinase+activity+reporter.">Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter.</a> Journal of Biological Chemistry. 282, 6733-6742 (2007).</li><li>Harvey, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+sensor+of+ERK+activity.">A genetically encoded fluorescent sensor of ERK activity.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 19264-19269 (2008).</li><li>Fosbrink, M., Aye-Han, N. N., Cheong, R., Levchenko, A., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+JNK+activity+dynamics+with+a+genetically+encoded+fluorescent+biosensor.">Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5459-5464 (2010).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+activation+of+CyclinB1-Cdk1+coordinates+entry+to+mitosis.">Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis.</a> Dev. Cell. 18, 533-543 (2010).</li><li>Fuller, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midzone+activation+of+aurora+B+in+anaphase+produces+an+intracellular+phosphorylation+gradient.">Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient.</a> Nature. 453, 1132-1136 (2008).</li><li>Ni, Q., Titov, D. V., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+kinase+dynamics+in+living+cells+with+FRET+reporters.">Analyzing protein kinase dynamics in living cells with FRET reporters.</a> Methods. 40, 279-286 .</li><li>Morgan, D. O. <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 cell cycle : principles of control. , (2007).</li><li>Lindqvist, A., Rodriguez-Bravo, V., Medema, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+decision+to+enter+mitosis:+feedback+and+redundancy+in+the+mitotic+entry+network.">The decision to enter mitosis: feedback and redundancy in the mitotic entry network.</a> J. Cell. Biol. 185, 193-202 (2009).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+cyclin+B1-Cdk1+synchronizes+events+in+the+nucleus+and+the+cytoplasm+at+mitosis.">Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis.</a> J. Cell. Biol. 189, 247-259 (2010).</li><li>Macurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polo-like+kinase-1+is+activated+by+aurora+A+to+promote+checkpoint+recovery.">Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.</a> Nature. 455, 119-123 (2008).</li><li>van der Eb, A. J., Graham, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+transforming+activity+of+tumor+virus+DNA.">Assay of transforming activity of tumor virus DNA.</a> Methods. Enzymol. 65, 826-839 (1980).</li><li>Lamprecht, M. R., Sabatini, D. M., Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+free,+versatile+software+for+automated+biological+image+analysis.">CellProfiler: free, versatile software for automated biological image analysis.</a> Biotechniques. 42, 71-75 (2007).</li><li>Roszik, J., Lisboa, D., Szollosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intensity-based+ratiometric+FRET+in+image+cytometry--approaches+and+a+software+solution.">Evaluation of intensity-based ratiometric FRET in image cytometry--approaches and a software solution.</a> Cytometry A. 75, 761-767 (2009).</li></ol>

我々は、開示することは何もない。

細胞周期を通してFRETを監視することで外部からの刺激に短期的応答を評価する際には、あまり重要な考慮事項が必要です。最初に、細胞周期の進行が容易にその光毒性を最小限にとどめる必要、ストレスのシグナリングによって摂動されています。第二に、すべての記者たちが潜在的にキナーゼ、ホスファターゼまたは相互作用ドメインを滴定することによって細胞プロセスに影響を与える...

<table border="1"><tbody><tr><td> 試薬 </td><td> カタログ番号 </td><td> 会社 </td></tr><tr><td>リーボビッツL - 15、無フェノールレッド</td><td> 21083-027 </td><td> GIBCO、Life Technologies社によって</td></tr><tr><td> DMEM +グルタミン- I </td><td> 31966 </td><td> GIBCO、Life Technologies社によって</td></tr><tr><td>ウシ胎児血清（FBS） </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0.05％トリプシンEDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO、Life Technologies社によって</td></tr><tr><td>ピューロマイシン</td><td> P8833 </td><td>シグマアルドリッチ</td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

フェルスター共鳴エネルギー移動（FRET）ベースの記者1は、生きた細胞中の内因性キナーゼとホスファターゼ活性の評価を可能にする。このようなプローブは、一般的にリン酸化可能なシーケンスとリン結合ドメインによる介入CFPとYFPの変異体、で構成されています。効率（図1）、FRETの変化につながる、CFPとYFPの間の距離や方向の変化をもたらすリン酸化の際、プローブの変更のコンフォメーション、。いくつかのプローブは、PKA 2、PKB 3、PKC 4、PKD 5、ERK 6、JNK 7、CDK 18、オーロラB 9の記者とPLK1 9を含む複数のキナーゼとホスファターゼの活性のバランスを、監視し、過去10年間の間に発表されている。モジュラー設計を考えると、追加のプローブは、近い将来10で出現するものと予想される。 細胞周期の進行は、ストレスsignaliによって影響されるngは11パスウェイ。特に、細胞周期が細胞周期を通じて細胞の細胞がストレスを12から回復している。タイムラプスイメージングでは、したがって、特に注意を必要とする場合に比べて成長を摂動の間に異なる規制されています。レシオメトリックイメージングを採用する場合は特に対ノイズ比の高い信号を持つ2つのイメージが正しく結果を解釈するために必要とされるので、これは、問題になります。キナーゼとホスファターゼ活性で細胞周期依存性変化のレシオメトリックFRETのイメージングは、主に細胞周期8,9,13,14のサブセクションに限定されています。 ここで、我々は、人間の細胞周期を通してレシオイメージングを用いたFRETに基づくプローブを監視する方法について説明します。方法は、ライフサイエンスにおける多くの研究者に利用可能であり、顕微鏡や画像処理の専門知識を必要としない機器に依存しています。

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRETに基づく記者はますます、生細胞におけるキナーゼとホスファターゼの活動を監視するために使用されています。ここでは、ターゲットのリン酸化の細胞周期依存性の変化を評価するためのFRETに基づくレポーターを使用する方法の方法を説明します。

FRETはレシオメトリックで細胞周期のキナーゼおよびホスファターゼの活動を監視

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

14.2K ビュー.  Karolinska Institutet. FRETに基づく記者はますます、生細胞におけるキナーゼとホスファターゼの活動を監視するために使用されています。ここでは、ターゲットのリン酸化の細胞周期依存性の変化を評価するためのFRETに基づくレポーターを使用する方法の方法を説明します。

ビデオ: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

伏在静脈を通って心臓穿刺によりラットから採血

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

生物学

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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 ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

1分子FRETによるDNAループを研究

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

の同期<em&gt;カウロバクターCrescentus</em&gt;細菌細胞周期の調査のため

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

同期を必要とせずに、フローサイトメトリーを用いた細胞周期の進行の時間追跡

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

ミトコンドリア局在と原子力細胞周期キナーゼ、Cdk1のの機能を研究するための実験的アプローチ

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

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, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

ライブ細胞懸濁液の蛍光光度アッセイでFRETセンサーを用いて測定したGタンパク質選択的なGPCRの立体配座

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...