オーストラリア原子科学技術学会のサポートが認められています。 TCKはAINSE賞を受賞しました。エピ医学研究所は、国立保健オーストラリアの医学研究評議会（566559）でサポートされています。 LMは、メルボルン研究所（メルボルン大学）と医学のCRCの補助金によってサポートされています。モナッシュマイクロイメージング（博士スティーブンコーディとISKAカーマイケル）のサポートは、この仕事のための非常に貴重でした。 MMTは、国立衛生研究所から博士オルガSedelnikovaから専門家の援助とアドバイスに感謝しています。

組織<ol><li>最適な切削温度（OCT）コンパウンド4583で金型に組み込まれた肺組織は、、CMライカ1950クライオスタットを用いて、ポリ- L -リジンスライド上にマウントされている（5μm）を切片であった。 </li></ol>凍結サンプルは、マードック児童研究所で博士サイモンロールスロイスから得られた。肺組織は、未処理のBalb / cマウス由来とヒトの喘息6の病理学的特徴の多くを示す慢性アレルギー性気道疾患のマウスモデルから得られた。動物実験は科学的な目的のために実験動物の管理と利用の実践のためのオーストラリアのコードに準拠しているマードック児童研究所の動物倫理委員会によって定められたガイドラインおよび規制に準拠して行った。 <ol start="2"><li>セクションは、1時間室温で風乾させた。 </li></ol>免疫蛍光染色<ol><li>セクションは、2％（w / v）のリン酸のパラホルムアルデヒドで20分間緩衝生理食塩水（PBS）で固定した。 </li><li>のセクションでは、15分ごとにPBSで2回洗浄と平衡化した。 </li><li>セクションは10分ごとに、PBSで3回洗浄した20分間70％エタノール（-20℃に冷却）で処理した。 </li></ol>この時点で、スライドは最大2週間まで4℃で密閉容器で保存することができる。 <ol start="4"><li>セクションは10分ごとに、PBSで3回洗浄し、次いで新鮮PBS含む0.5％のTween - 20及び0.1％トリトンX - 100室温で1時間（PBS - TT）の8％BSAを含むPBSで調製された100μLでブロックされた。 すべてのインキュベーションは加湿染色トラフで実施した。 </li><li>セクションは、5分間PBS - TTで洗浄し、疎水性の境界は、パパニコロウペンを持つ組織の周囲にマークされました。 </li><li>過剰なバッファがオフチップを渡したされ、一次ウサギポリクローナル抗γH2AX抗体（PBS中の1％BSAで、1:500に希釈し、ミリポア）100μlのは、室温で2時間のインキュベーションのために各セクションに追加されました。 一次抗体とのインキュベーションを4℃で一晩実施されることがあります さらなる証拠を、γH2AX病巣がDSBのを表していること、DSBの修復のタンパク質を認識する一次抗体を使用してもよい。例えば、γH2AXとリン酸化ATMの共局在は、一般的に使用される組み合わせです。 </li><li>暗所で室温で1時間、セクションが各5分間PBS - TTで2回洗浄し、二次抗体液100μl（Invitrogen社のAlexa Fluor ® 488ヤギ抗ウサギIgGを1％BSAで、1:500に希釈）とともにインキュベートした。 （希釈した抗体は、プロシージャ全体で暗所で保管された）。 </li><li>セクションは、各5分間PBS - TTで3回洗浄し、37℃で30分のため0.5mg/mL RNアーゼA℃でインキュベートした。 </li><li>対比と取り付けの核はヨウ化プロピジウムを含むVectashieldの培地で行った。 </li><li>スライドはマニキュアで密封し、℃の暗所での分析の前に4℃で一晩保存した。 </li></ol>顕微鏡/分析<ol><li> （ - のAlexa Fluor ® 488ヤギ抗ウサギIgGγH2AXのため）とPI（543 nm）のレーザーツァイスLSM510のメタ共焦点顕微鏡は、標準的なGFPを使用して画像を取得するために使用されていました。 </li></ol>画像は、0.5μmのステップサイズとZシリーズのパターンで獲得されました。分析中に、個々の面は、デコンボリューションと巣の重複を最小限に抑えるために最大限の投影画像を生成するために積層した。 前の粒子数の最大値投影画像を作成する方法は、薄切片（例えば、5μm以下）を使用し、バックグラウンド染色はわずかですどこにされる状況でのみ使用する必要があります。ここで、重要なバックグラウンドを持つ厚いセクションがBolteとCordelieresで説明されているように、このような3Dオブジェクトカウンタなど、より複雑な分析を分析する必要があります（2006年）7を使用する必要があります。 <ol start="2"><li> Metamorph（Molecular Devices社、米国）は、病巣の数を定量するためにマージ（赤と緑）の画像を作成するために使用されました。 </li></ol> Metamorphが巣の数を定量化するために使用できますが、組織切片を使用して、通常、時病巣は、より高い精度のために（目で）手動でカウントされます。 興味の特定の細胞型は、インスタンスの肺上皮細胞のために、定量用に選択することができます。これは、組織学上及びヘマトキシリン​​及びエオシン染色連続切片への参照を持つ基づいて行われることがあります。

<ol>
	<li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+H2AX+and+cancer.">Gamma H2AX and cancer.</a> Nature Reviews Cancer. 8 (12), 957-967 (2008).</li><li>Dickey, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. 118 (6), 683-692 (2009).</li><li>Bhogal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+residual+gamma-H2AX+foci+in+murine+skin+are+dose+responsive+and+predict+radiosensitivity+in+vivo.">Late residual gamma-H2AX foci in murine skin are dose responsive and predict radiosensitivity in vivo.</a> Radiat Res. 173 (1), 1-9 (2010).</li><li>Redon, C. E., Dickey, J. S., Bonner, W. M., Sedelnikova, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-H2AX+as+a+biomarker+of+DNA+damage+induced+by+ionizing+radiation+in+human+peripheral+blood+lymphocytes+and+artificial+skin.">gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin.</a> Adv Space Res. 43 (8), 1171-1178 (2009).</li><li>Locke, N. R., Royce, S. G., Wainewright, J. S., Samuel, C. S., Tang, M. L., L, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+airway+remodeling+in+acute,+subacute,+and+chronic+models+of+allergic+airways+disease.">Comparison of airway remodeling in acute, subacute, and chronic models of allergic airways disease.</a> Am J Respir Cell Mol Biol. 36 (5), 625-632 (2007).</li><li>Bolte, S. &. a. m. p. ;. a. m. p., Cordelieres, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> J Microsc. 224 (Pt 3), 213-232 (2006).</li></ol>

利害の衝突は宣言されません。

このデモンストレーションの目的のために我々は、未処理のBalb / cマウスからと慢性アレルギー性​​気道疾患のマウスモデルからマウスの肺組織を使用する。我々は、未処理のセクションに比べて損傷した肺で観察されたわずかに高い平均で細胞あたりの巣の数の違いを定量。特に、定量はそれぞれ、未処理組織と慢性アレルギー性​​気道疾患の組織で、/セルあたり6.5巣および/セルあた...

<table><tbody><tr><td>Bovine Serum Albumin (BSA)</td><td></td><td>Sigma-Aldrich</td><td>A7906</td><td>BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.</td></tr><tr><td>PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td></td><td>Invitrogen</td><td>17-517Q</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound 4583</td><td></td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Superfrost Plus</td><td>Polysine slides</td><td>Menzel-Glaser</td><td>SF41296SP</td><td>Electrostatically attracts frozen tissue sections and reduces the need for additional coatings and adhesives.</td></tr><tr><td>Tween 20</td><td>Reagent</td><td>Calbiochem</td><td>655205</td><td>Tween 20 (0.5%), is a detergent to permeabilise cells</td></tr><tr><td>Triton X-100</td><td>Reagent</td><td>Sigma-Aldrich</td><td>T8787</td><td>Triton X-100 (0.1%) used to permeabilise cells.</td></tr><tr><td>Paraformaldehyde</td><td>Reagent</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Anti-phospho-&amp;#947;H2AX (rabbit polyclonal antibody)</td><td>Primary Antibody</td><td>Upstate, Millipore</td><td>07-164</td><td>Dilution of primary antibody (1:500), in 1% BSA.</td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td><td>Secondary Antibody</td><td>Invitrogen</td><td>A-11008</td><td>Dilution of secondary antibody (1:500), in 1% BSA.</td></tr><tr><td>Ethanol (70%)</td><td></td><td>Thermo Fisher Scientific, Inc.</td><td>BSPEL316</td><td></td></tr><tr><td>RNAse A</td><td>Reagent</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Vectashield PI</td><td>Reagent</td><td>Vector Laboratories</td><td>H-1300</td><td>A glycerol based mounting medium containing propidium iodide (a red nuclear stain).This medium must be used with an oil based lense that matches its refractive index.&lt;br/&gt;&lt;br/&gt;Note: DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.</td></tr><tr><td>Mini PAP Pen</td><td></td><td>Invitrogen</td><td>008877</td><td></td></tr><tr><td>Coverslips (22x50mm)</td><td></td><td>Menzel-Glaser</td><td>CS2250100</td><td></td></tr><tr><td>Coplin Jar, glass</td><td></td><td>Grale Scientific P/L</td><td>1771-OG</td><td></td></tr><tr><td>Staining Trough</td><td></td><td>Grale Scientific P/L</td><td>V1991.99</td><td></td></tr><tr><td>Zeiss LSM 510 Meta Confocal</td><td></td><td>Confocal Microscope</td><td></td><td></td></tr><tr><td>CM Leica 1950 Cryostat</td><td></td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Metamorph</td><td>Software for Imaging analysis</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

quantitation of &gamma;h2ax foci in tissue samples

DNA二本鎖切断（DSB）は、内因性（生理学的または病理学的）プロセスによって、または外因性の因子、特に電離放射線と放射線作用の化合物のいずれかによって発生する可能性、特に致命的と遺伝毒性病変、です。セリン- 139残基が、高度に保存されたC末端SQEYのモチーフで、γH2AXを形成し、DNA二本鎖切断に早い応答であるにH2Aヒストンバリアント、H2AXのリン酸化<sup&gt; 1</sup&gt;。このリン酸化のイベントは、ホスファチジル - inosito 3 - キナーゼ（PI3K）タンパク質のファミリー、毛細血管拡張性運動失調変異（ATM）、DNA -プロテインキナーゼ触媒サブユニットとATMとRAD3関連によって媒介される（ATR）<sup&gt; 2</sup&gt;。全体的に、離散的な核γH2AX巣の形成におけるDSBの誘導の結果は容易に検出し、免疫蛍光顕微鏡法で定量することができる<sup&gt; 2</sup&gt;。このマーカーのユニークな特異性と感度を考えると、γH2AX巣の分析は、特に、放射線生物学、核医学では、生物医学研究の幅広い用途につながっている。 γH2AX病巣の定量は、最も広く電離放射線誘発DSBがコンテキスト内で細胞培養系で検討されている。離れて細胞放射線感受性から、免疫蛍光ベースのアッセイはまた、放射線改質化合物の有効性を評価するために使用されています。さらに、γH2AXは様々なDSBを誘発する化合物の有効性を検討するための分子マーカーとして使用されており、最近では老化や病気の重要なマーカー、特にがんとして歓迎されている<sup&gt; 3</sup&gt;。さらに、免疫蛍光ベースの方法はγH2AX巣の検出および定量に合うように適応されている<em&gt;生体外で</em&gt;と<em&gt; in vivoで</em<sup&gt; 4,5</sup&gt;。ここで、我々はマウス組織におけるγH2AX巣の検出および定量するための典型的な蛍光抗体法を示しています。

Watch this Scientific Journal Video about Quantitation of Î³H2AX Foci in Tissue Samples at JoVE.com

Quantitation of &#947;H2AX Foci in Tissue Samples

γH2AX巣に基づいてDNA二本鎖切断の定量は、組織の放射線感受性と放射線修正する化合物の作用の評価に、特に放射線の生物学では、非常に貴重なツールとなっています。ここでは、組織サンプルにおけるγH2AX病巣の定量のための免疫蛍光アッセイの使用方法を示しています。

組織サンプルのγH2AX病巣の定量

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

quantitation-of-h2ax-foci-in-tissue-samples

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

14.3K ビュー.  The Alfred Medical Research and Education Precinct. γH2AX巣に基づいてDNA二本鎖切断の定量は、組織の放射線感受性と放射線修正する化合物の作用の評価に、特に放射線の生物学では、非常に貴重なツールとなっています。ここでは、組織サンプルにおけるγH2AX病巣の定量のための免疫蛍光アッセイの使用方法を示しています。

ビデオ: Quantitation of γH2AX Foci in Tissue Samples

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

ΓH2AX、53BP1 形成と DNA 二重鎖切断の修復を分析するための蛍光顕微鏡観察

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

がん研究

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

蛍光顕微鏡を用いた過渡的長期的な二本鎖 DNA 切断の DNA 修復プロセスの特性

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

フローサイトメトリーによる無毒ストレス後のγH2AXとアポトーシスの細胞周期特異的測定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

ヒト大腸癌細胞におけるDNA損傷および修復病巣の免疫蛍光イメージング

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

ヒト末梢血リンパ球におけるγ-H2AX Fociアッセイの自動微視的スコアリング法

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

ヒト末梢リンパ球におけるγH2AXと53BP1の二重免疫蛍光法

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

遺伝学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

生物学

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

電離放射線は、次のγH2AXの空間分布の評価

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

誘導および定量化細胞老化するテクニック

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.