この作品は、国内機関の健康の付与 (CA206157-22 と GM118066) とアンジェリカ アモンにぞんざいな大理石がん研究基金によってコッホ研究所サポート (コア) グラント P30 CA 14051 国立がん研究所から一部支持されました。アンジェリカ アモンもハワード ヒューズ医学研究所と医歯薬学研究グレン財団法人の捜査官です。アメリカ イタリアがん財団 (AICF)、マリー ・ キュリー ・ アクションから癌研究 (AIRC) のためのイタリアの協会がん研究の交わりによって、キ Quinquennial がん研究員、s. s. がサポートされていました。E. m. は、ハワード ヒューズ医学研究所から奨学金によって支えられました。

培養条件
RPE 1 テロメラーゼ セルラインだったダルベッコ変更イーグル培地中で培養 (材料表) 10% を添加したウシ胎児血清、2 mM L-グルタミン 100 U/ml ペニシリン/ストレプトマイシン。セル 5% CO2加湿環境で 37 ° C で培養。下記プロトコル 10 cm 皿を使用して開発されているし、報告されるすべての技術的な詳細は、これらの料理を参照してください。別の料理を使用している場合スケール アップまたはそれに応じてダウンします。
1. 網膜色素上皮細胞の同期
<ol><li>すべての実験のための以上 15 通路を受けていない新鮮な解凍のセルを使用します。1 日に解凍し、~2.5 - 10 cm 培養皿に 105 RPE 1 テロメラーゼ セル x 5.0 を調剤します。標準的な成長媒体の 8 mL を追加し、一晩インキュベートします。(診断) などの標準的な細胞カウンターを使用してセル数を決定します。 注: セル密度は RPE 細胞の成長条件を指します。成長条件が異なるセルは並ぶ; の間で異なる場合があります。それらをそれに応じて調整することを検討してください。</li>
	<li>2 日目、通常成長培地を吸引し、5 mM チミジンを含む 8 mL の培地を追加して G1/S のボーダーでセルを同期します。 注: チミジン濃度と治療期間を異なる細胞間で異なる場合があります。テストするセルの行の最適な条件を決定するためのパイロット実験を実行することをお勧めします。</li>
	<li>3 日目、24 h チミジン培地 (ステップ 1.2) を追加した後、培地を吸引・洗浄の 3 回 1 × PBS のセルによってチミジン ブロックからのリリースします。通常培 8 mL を追加し、インキュベーターにプレートを返します。</li>
</ol>2 世代異数性の細胞分裂期キナーゼ Mps1 の活動と干渉
<ol><li>3 日目、リリース後 6 h チミジン ブロックから (ステップ 1.3; 細胞が有糸分裂を入力する前に、これは約 3-6 時間)、培地を吸引、1 × PBS で 1 回洗浄し、500 nM リバーシン (Mps1 阻害剤) を添加した培地で置き換えます。 注: RPE 細胞有糸分裂 9 12 h を入力後チミジン ウォッシュ アウト10,11。ただし、細胞周期は異なる細胞株によって異なります。したがって、(例えば、DNA コンテンツを介してFACS 解析を測定することによって) 確立された方法によって細胞周期解析を実行してパイロット実験におけるそれらの動態を決定することが重要です。さらに、細胞間最適な作業 Mps1 阻害剤濃度が異なる場合があります。RPE1 細胞を用いた以前の実験は、500 nM10、11,12の作業濃度リバーシンを使用して成功しています。テストするセルの行の最適な濃度を決定するためのパイロット実験を実行することをお勧めします。</li>
	<li>日目 4、12 h リバーシン治療 (約 6 h 細胞有糸分裂後) 後、リバーシン培地を吸引し、洗浄 3 回 1 × PBS のセル。プレートに 8 mL の正規の成長培地を追加し、インキュベーターにセルを返します。</li>
</ol>3. Aneuploid サイクリング細胞と ArCK 人口の濃縮の除去
<ol><li>日 6、約 72 時間細胞を入力最初の細胞分裂後に (手順 2.2; リバーシン洗浄アウト後, 約に相当する約 66 時間は、この 2-3 細胞周期)、培地を吸引、1 × PBS で 1 回洗浄、300 nM ノコダゾールを含有する培地に置き換えます。 注: 最近の仕事は示した RPE 1 細胞の染色体の異数性 genomically 不安定について彼らの増殖を逮捕最初不良有糸分裂10の後の 2-3 細胞周期。ただし、この速度は異なる細胞の間で異なる場合があります。したがって、構成するサイクリング細胞の除去のための適切なタイミングを決定するためのパイロット実験を実行することが重要です。</li>
	<li>一日 7、ノコダゾール治療後 12 h 培地を吸引し、プレートに 3 mL の 1x PBS を追加します。プレートの側面をタップすることで分裂期の細胞を振り払います。分裂細胞も除去できるように各タップ プレートを回転させます。</li>
	<li>分裂期細胞の完全な除去を確実には、複数のラウンド (3-5 回を推奨)、吸引・追加 3 mL の 1x PBS 各ラウンド間の振り払いプロセスを繰り返します。</li>
	<li>振り払い後、軽く蓋または板のエッジに付着した液体を吸い出しなさい。簡単に言えば、10 倍の倍率の顕微鏡の下でプレートを確認してください。いくつかの分裂期の細胞が観察されることを確認します。5 つ以上の分裂期の細胞は、対物レンズ 10 倍の下で見られている、振り払いの別のラウンドを繰り返します。 注: 分裂期細胞は丸みを帯びているが、振り払い後部分的に切断可能性があります。ノコダゾール治療の次のラウンドに細胞を公開する前に分裂細胞の完全な除去のために重要です。そう失敗はおよび/または四倍体細胞生成の結果有糸分裂細胞死につながる可能性があります。</li>
	<li>最終的な揺れオフ後 5 mL の 1x PBS で 1 回細胞を洗います。プレートに 330 nM ノコダゾールを含む 8 mL の培地を追加し、12 時間インキュベートします。</li>
	<li>孵化後分裂細胞が観察されなくなるまでノコダゾール治療/振り払い (ステップ 3.1 3.5) 12 時間間隔を繰り返します。通常、治療/手ふれ off の 4-5 回を推奨します。 注: ここで提供される情報は、RPE 細胞を参照してください。治療/手ふれ off のラウンド数は、異なる細胞間で異なる場合があります。ノコダゾールに長引くと不必要な露出を避けるために、慎重に判断して、パイロット実験における異数サイクリング細胞の効率的かつ完全な除去のために必要なラウンド数に不可欠です。</li>
	<li>ノコダゾール治療/振り払い (ステップ 3.1 3.6) の 4 〜 5 ラウンドの後通常成長培地 10 mL を加えるし、孵化させなさい。将来の操作または試金の使用前に 12-24 時間の残りの部分にセルを許可します。 注: 最終振るオフ後セルが皿に残って逮捕細胞周期、最近10、示すように、複雑な染色体を抱きます。これらの細胞は、弧 (複雑核型で逮捕) 細胞と呼ばれます。分離の一週間以内 ArCK 集団でアッセイを実行することをお勧めします。</li>
	<li>必要に応じて、複雑な染色体で逮捕された細胞の割合を評価するために収集し、スタンダード セルのカウンターを使用して各シェイクオフからセルをカウントします。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg"> 図 1: 生成し、複雑核型 (弧) と代表的な画像を構成する細胞の分離に使用するメソッドの全体的なスケマティック。(A)図の ArCK 細胞世代と分離のプロトコル。(B) RPE 1 テロメラーゼ細胞治療の各段階での代表的なイメージ。(赤で記載) の最後のパネルでは、分離の ArCK セルを表します。スケール バー 100 μ m.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
4. ArCK 人口の特性評価
<ol><li>次の試金の実行によって ArCK 人口の分離を成功させるを確認します。<ol>
<li>ロバ β-ガラクトシダーゼ (老化マーカー) を染色、市販を使用してキット (材料の表を参照してください)。</li>
		<li>市販キット (材料の表を参照) を用いて CCL2 などの炎症性サイトカインの分泌を測定します。</li>
		<li>細胞周期阻害剤 p53、p21、ウェスタンブロットで p16 の増加されたレベルを確認します。 注: これらのアッセイを正しく制御するため ArCK 集団は欠異数のサイクリング細胞と比較する必要があります。前者は低通過、野生型 RPE 1 テロメラーゼ細胞は指数関数的に成長しています。後者は RPE 1 テロメラーゼの偏析を誤染色体を誘導する、最初異常有糸分裂後 72 時間のセルを収穫して取得できます。これを行うには、最初のノコダゾール治療 (ステップ 3.1) の前に右ステップ 1.1 および収穫細胞から上記プロトコルに従います。</li>
	</ol>
</li>
	<li>逮捕された細胞の核型を評価するために単一セルのシーケンスを実行します。 メモ: 単一セルの配列は、単細胞レベル13でセル人口の間で染色体とサブ染色体の変化を調べること確立された方法です。単一セルのシーケンスを実行する方法の詳細な手順は、他の場所で10で見つけることができます。</li>
</ol>

<ol><li>Santaguida, S., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-+and+long-term+effects+of+chromosome+mis-segregation+and+aneuploidy%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Short- and long-term effects of chromosome mis-segregation and aneuploidy.</a> Nature Reviews Molecular Cell Biology. 16 (8), 473-485 (2015).</li>
<li>Pfau, S. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+instability+and+aneuploidy+in+cancer%3A+from+yeast+to+man%5BTitle%5D)+AND+%22EMBO+reports%22%5BJournal%5D)">Chromosomal instability and aneuploidy in cancer: from yeast to man.</a> EMBO reports. 13 (6), 515-527 (2012).</li>
<li>Gordon, D. J., Resio, B., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Causes+and+consequences+of+aneuploidy+in+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Causes and consequences of aneuploidy in cancer.</a> Nature Reviews Genetics. 13 (3), 189-203 (2012).</li>
<li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Boveri+revisited%3A+chromosomal+instability%2C+aneuploidy+and+tumorigenesis%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nature Reviews Molecular Cell Biology. 10 (7), 478-487 (2009).</li>
<li>Meena, J. K., Cerutti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomerase+abrogates+aneuploidy-induced+telomere+replication+stress%2C+senescence+and+cell+depletion%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.</a> The EMBO Journal. , 1-14 (2015).</li>
<li>Ohashi, A., Ohori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+generates+proteotoxic+stress+and+DNA+damage+concurrently+with+p53-mediated+post-mitotic+apoptosis+in+SAC-impaired+cells%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.</a> Nature Communications. 6, 7668(2015).</li>
<li>Passerini, V., Ozeri-Galai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+presence+of+extra+chromosomes+leads+to+genomic+instability%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">The presence of extra chromosomes leads to genomic instability.</a> Nature Communications. 7, 10754(2016).</li>
<li>Sheltzer, J. M., Blank, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+drives+genomic+instability+in+yeast%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Aneuploidy drives genomic instability in yeast.</a> Science. 333 (6045), 1026-1030 (2011).</li>
<li>Zhu, J., Pavelka, N., Bradford, W. D., Rancati, G., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Karyotypic+determinants+of+chromosome+instability+in+aneuploid+budding+yeast%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Karyotypic determinants of chromosome instability in aneuploid budding yeast.</a> PLoS Genetics. 8 (5), e1002719(2012).</li>
<li>Santaguida, S., Richardson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+Mis-segregation+Generates+Cell-Cycle-Arrested+Cells+with+Complex+Karyotypes+that+Are+Eliminated+by+the+Immune+System%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.</a> Developmental Cell. 41 (6), 638.e5-651.e5 (2017).</li>
<li>Santaguida, S., Vasile, E., White, E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy-induced+cellular+stresses+limit+autophagic+degradation%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Aneuploidy-induced cellular stresses limit autophagic degradation.</a> Genes & Development. 29 (19), 2010-2021 (2015).</li>
<li>Santaguida, S., Tighe, A., D'Alise, A. M., Taylor, S. S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+the+role+of+MPS1+in+chromosome+biorientation+and+the+spindle+checkpoint+through+the+small+molecule+inhibitor+reversine%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.</a> The Journal of Cell Biology. 190 (1), 73-87 (2010).</li>
<li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genome+sequencing%3A+current+state+of+the+science%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175-188 (2016).</li>
<li>López-Soto, A., Gonzalez, S., López-Larrea, C., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunosurveillance+of+Malignant+Cells+with+Complex+Karyotypes%5BTitle%5D)+AND+%22Trends+in+cell+biology%22%5BJournal%5D)">Immunosurveillance of Malignant Cells with Complex Karyotypes.</a> Trends in cell biology. , 1-4 (2017).</li>
<li>Watson, E. V., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+Police+Detect+Chromosomal+Imbalance+Triggering+Immune+Crackdown%21%5BTitle%5D)+AND+%22Trends+in+genetics%3A+TIG%22%5BJournal%5D)">Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown!</a> Trends in genetics: TIG. , 1-3 (2017).</li>
</ol>

著者が明らかに何もありません。

この新規メソッドを生成し、複雑な染色体 (弧) の逮捕された電池の豊かは、複数の染色体の利得または損失を持ち、分割停止細胞の研究のためことができます。方法セットアップは迅速かつ信頼性の高い方法で ArCK 細胞の分離を容易にする設計されています。
この試験で最も重要なステップは、薬物治療のタイムラインと、最も重要なは、構成するサイクリング細胞の除去厳密制御です。最適な結果を得るのためのノコダゾール治療と振るオフのタイミングは特に重要な彼らはまだ丸みを帯びたと分裂、G1 に分裂細胞死やスリッページの可能性を防止するプレートからサイクリングのセルを削除するには四倍体の人口を作成する可能性があります。推奨 12 h ノコダゾール インキュベーションから 2 時間以上を振り払いのタイミングに逸脱はお勧めできません。
将来に関する ArCK 細胞どのように複雑な染色体のより深い理解を促進する可能性がある細胞生理学に影響を与えます。特に、最近の研究は、免疫系の細胞と対話すること、トリガー免疫クリアランス ArCK の細胞10を示した。ここで述べる方法免疫クリアランス未変換細胞とどのように発癌の研究の基礎となる分子メカニズムの解明を含む ArCK セルのさらなる評価のため優秀な出発点を提供します。この監視のメカニズム、フィールド14,15の重大な質問をバイパスがあります。

染色体分配の過程でエラーは、異数性半数体補1,2,3,4の倍数ではない染色体数によって特徴付けられる条件に します。異数性染色体ゲノム不安定性5,が生成されますさらにトリガーのレプリケーション ストレス6,7,8,9,10増加核型の複雑さ、および最終的に細胞10個体の細胞周期の停止につながる。ここで紹介した方法の目的を生成し細胞をサイクリングからこのような個体を分離です。安価な培養技術を採用し、このプロトコル分離と複雑な染色体の異数性細胞の細胞周期逮捕の評価が容易になります。これらの細胞は、ArCK (複雑核型で逮捕) セルおよびコントロールとしてサイクリング欠の相手として呼ばれます。
このプロトコルは、この目的のために確立される最初の 1 つを分離することができ、さらなる研究の ArCK 線を含む、彼らの誘導老化と老化関連の分泌形質 (SASP) に限定されない、プロ炎症機能、および能力を免疫細胞に関与します。ここで紹介した方法未変換、不死化ひと細胞で開発されていますが、がんでまだテストされていません。いくつかの変換されたセルが 1 つまたは複数の経路の抑制による細胞周期の停止に敏感である可能性があります。さらにしたがって、他の細胞では検証を実行する必要があります。

<table><tbody><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle Medium)&amp;nbsp;</td><td>Thermofisher</td><td>11995-073</td><td></td></tr><tr><td>Thymidine</td><td>Sigma Aldrich</td><td>T1859</td><td></td></tr><tr><td>Reversine</td><td>Cayman Chemical Company</td><td>10004412</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma Aldrich</td><td>M1410</td><td></td></tr><tr><td>Anti-p53 antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Anti-p21 antibody</td><td>Santa Cruz</td><td>sc-6246</td><td></td></tr><tr><td>Anti-p16 antibody</td><td>BD</td><td>554079</td><td></td></tr><tr><td>Senescence b-Galactosidase Staining Kit</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9860</td><td></td></tr><tr><td>Proteome Profiler Human Cytokine Array Kit&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>ARY005B</td><td></td></tr></tbody></table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

染色体の誤った分離は、異数性、細胞が不均衡な染色体数を港を条件に します。証拠の複数のラインは強くその異数性をゲノムの不安定性は、最終的に彼らの増殖を阻止する複雑な染色体を持つ細胞を生成するトリガーを示します。細胞の複雑な染色体の分離と性状、細胞生理学的不均衡な染色体数の影響を検討することが重要です。日には、手法が確立されてないような構成する細胞を確実に分離します。このペーパーでは、標準の安価な培養技術を利用した複雑な染色体の濃縮と異数性細胞の解析のためのプロトコルを提供します。このプロトコルは、その誘導分泌老化関連の表現、プロ炎症性のプロパティ、および免疫細胞と対話する能力を含む複雑な染色体を構成する細胞のいくつかの機能を分析する使用することができます。がん細胞は、染色体数の不均衡をしばしば港、異数性が両方のプロと反 tumorigenic 効果を暴くの究極の目標と、正常細胞の細胞生理に及ぼす影響について解読することが重要です。

このメソッドは、彼らの増殖を阻止する複雑な染色体を構成する細胞を分離するための in vitro培養システムを利用しています。この人口は、ArCK (逮捕された複雑な染色体で) 細胞と呼ばれます。図 1 aは、実験の方法を示します。野生型サイクリング RPE 1 テロメラーゼ細胞チミジンと G1/S の国境で、同期化、染色体の誤った分離を誘発する Mps1 阻害剤と扱われます。スピンドルの毒ノコダゾールは、染色体の誤った分離の誘導後 72 時間のセルに追加されます。異数性の細胞増殖するまだことができるノコダゾール治療後 12 時間は有糸分裂を入力し、微小管重合の干渉のためには、この細胞周期段階に閉じ込められています。閉じ込められたこれらのセルが穏やかな振り払いによって簡単に削除されます。振り払いプロセスは繰り返し 3 - 5 回、サイクリング細胞を完全に除去です。図 1 bは、治療の各段階での RPE 細胞の代表的なイメージを示しています。
ArCK 細胞は、p53、p21、図 2 aに示すように、欠と異数性細胞をサイクリングに比べて p16 などの細胞周期阻害剤の増加されたレベルを表示します。また、図 2 bは、肯定的な β-ガラクトシダーゼ染色 ArCK 欠細胞と比較して細胞の細胞老化の十分に確立されたマーカーを示します。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg"> 図 2: ArCK 細胞を利用した代表的な結果。((A))の西部のしみ欠、サイクリング、異数性、ArCK 細胞の細胞周期阻害剤レベルを評価します。P53、p21、p16 のレベルは、西部のしみの分析により決定しました。アクチンはローディング コントロールとして提供しています。(B) β-ガラクトシダーゼ染色 ArCK 細胞老化のレベルを評価します。欠細胞と ArCK 細胞老化関連 β ガラクトシダーゼ (β Gal) 活動を求めた。スケール バー 100 μ m.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
この細胞周期の停止は、ランダム染色体の利得そして損失で図 3ArCK セルが複数表示されるシーケンスの単一セルから代表的な核型分析によって示されている複雑な染色体を構成する細胞の顕著な特徴です。この発見は、以前のレポート10との完全な合意です。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg"> 図 3: ArCK 細胞の代表的な単一セル配列。セグメンテーションは、六つの代表的な ArCK 細胞の核型を示すプロットされます。セグメント化プロットは、(RPE 1 野生型は女性の出自の 2 倍体細胞ライン) ログ2規模で欠基準 X に 1 からすべての染色体のコピー数が表示されます。染色体の利益は、緑色で、赤の染色体損失でハイライトされます。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

異数性は、ゲノムの不安定性、複雑な染色体を持つ細胞を細胞周期逮捕を最終的に生成に します。このペーパーでは、複雑な染色体を分割するために停止を構成する細胞を分離する簡単で便利な方法を提供します。

世代と複雑な染色体と細胞周期逮捕細胞の分離

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

generation-isolation-cell-cycle-arrested-cells-with-complex

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.4K ビュー.  Massachusetts Institute of Technology. 異数性は、ゲノムの不安定性、複雑な染色体を持つ細胞を細胞周期逮捕を最終的に生成に します。このペーパーでは、複雑な染色体を分割するために停止を構成する細胞を分離する簡単で便利な方法を提供します。

ビデオ: Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

2つの相補的細胞同期プロトコルによる細胞周期制御遺伝子発現の研究

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

遺伝学

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

遠心浄化によって異なった細胞周期の段階で主に急性リンパ性白血病細胞の調製

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

がん研究

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳類の細胞周期中に RNA 活性化蛋白質キナーゼの RNA インターアクターを勉強

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

生化学

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

細胞分裂時の染色体分配の生きているセルイメージ投射

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

発生生物学

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

染色体分析のためのヒト胚性幹細胞の染色体スプレッドの準備

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

培養細胞からの染色体の準備

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

無細胞アッセイは、有糸分裂の終了時クロマチン脱凝縮を勉強します

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...