Name: immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks
Uploaded: 2017-11-03T14:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Microscopy of ÃŽÂ³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

プロジェクトは、ドイツのホセ ・ カレーラス白血病財団 (DJCLS 14 R/2017) によって支えられました。

ここで説明したすべてのメソッドは、ハイデルベルク大学医療学部マンハイムの倫理委員会 II によって承認されています。インフォームド コンセントを得たすべての個人から書き込まれます。
 1。 材料準備 <ol><li>抗凝固剤原液: 0.9% 塩化ナトリウムの mL あたり 200 i. u. ヘパリンの抗凝固薬の原液を準備。コレクション チューブのそれぞれを入力 (ボリュー...

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ΓH2AX、53BP1 の蛍光顕微鏡は、形成と幅広い分野で DSB の修復の分析に有用な方法です。実験の結果に影響を与える重要なパラメーターは、細胞周期、固定と透過の細胞、抗体、およびハードウェアの選択、蛍光顕微鏡のソフトウェアに使用されるエージェントの段階です。
正常線維芽細胞細胞株培養を指数関数的に成長することにより細胞周期の γH2AX の巣パターンに及?...

DNA が継続的に内因性によって破損している (例えば、複製ストレス、活性酸素種、DNA の固有不安定性) と外因性 (例えば、化学ラジカル、照射) ソース (図 1)1,2 ,3,4。DNA 損傷の間で DNA 二重鎖切断 (DSB) は特に深刻な病変、細胞死やがんを引き起こす可能性があります。細胞と細胞周期5あたり約 50 DSB が生じることがあります。哺乳類細胞における相同組換え (HR) と非相同性末端結合 (NHEJ) は、DSB (図 2) の修理のための主要な経路として開発。HR 遅い S/G2 段階で発生し、そのまま妹を使用する可能性のあるエラー無料修理のためのテンプレートとして染色分け体。比較では、NHEJ は細胞周期の中でアクティブおよび潜在的変異原性塩基対を追加または壊れた端部の手術時に切除したことがあります。さらに、代替末端結合 NHEJ 欠乏6、7の場合遅い変異原性バックアップ修理メカニズムとして従事するかもしれない。
DSB は、各 DSB の周りいくつかの megabase ペアの領域でセリン 139 でヒストン H2AX のリン酸化を誘導します。形成の核の巣 γH2AX 巣の名前は、蛍光顕微鏡8で検出。ΓH2AX はさらに DSB 応答性タンパク質の採用を促進し、クロマチン DNA 修復、信号伝達9に関与しています。各 γH2AX フォーカスは、単一の DSB を表すと考えられている、蛍光顕微鏡による DSB の定量化が可能であれば、癌細胞および患者検体10、11,が示されています12,13,14. 53BP1 (p53 結合タンパク質 1) DSB 修復を媒介に別の主要蛋白質であります。DSB 応答性タンパク質、チェックポイントシグナ リング、DSB 端15のシナプスの募集に取り組んでいます。さらに、53BP1 は DSB 修復経路選択に重要な役割を果たしています。HR は防がれた16NHEJ に向かって DSB の修復がトリガーされます。ΓH2AX と DSB 修復 53BP1 の本物の機能を考慮した γH2AX と蛍光顕微鏡により 53BP1 の同時分析形成と DSB の修復の正確な分析のための有用な方法があります。
この原稿は、細胞核の γH2AX、53BP1 の蛍光顕微鏡を実行するためのステップバイ ステップ プロトコルを提供します。具体的には、細胞株培養、CD34 陽性急性骨髄性白血病の患者の細胞、健康な人の x 線照射によるリンパ球の正常線維芽細胞に応用されました。方法の詳細は、発表された結果のコンテキストで指摘されます。

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">RPMI medium</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">R0883</td>
			<td style="width:200px;">Medium for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Heparin sodium</td>
			<td style="width:119px;">ratiopharm</td>
			<td style="width:104px;">PZN 3029843</td>
			<td style="width:200px;">&#160;Heparin 5,000 I.U. / mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium chloride solution 0.9%</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:104px;">PZN 1957154</td>
			<td style="width:200px;">Component of the anticoagulant stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Ficoll-Paque Premium</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:104px;">17-5442-02</td>
			<td style="width:200px;">Medium for isolation of mononuclear cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Trypsin solution 10X</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">59427C</td>
			<td style="width:200px;">Enzyme for dissociation of fibroblasts in cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">CD34 MicroBead Kit</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-046-702</td>
			<td style="width:200px;">Isolation of CD34+ myeloid progenitor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Diagnostic microscope slides</td>
			<td>Thermo Scientific</td>
			<td>ER-203B-CE24</td>
			<td style="width:200px;">Microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Megafuge 1.0 R</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003060</td>
			<td style="width:200px;">&#160;Tabletop centrifuge&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cytospin device</td>
			<td style="width:119px;"></td>
			<td style="width:104px;"></td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Lid</td>
			<td style="width:119px;">Heraeus</td>
			<td>76003422</td>
			<td style="width:200px;">Lid for working without micro-tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cyto container</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003416</td>
			<td style="width:200px;">Cyto container with 2 conical bores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Clip carrier</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003414</td>
			<td style="width:200px;">Carrier for holding a cyto container and a slide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Support insert</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003417</td>
			<td style="width:200px;">Support insert for holding a clip carrier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Triton X-100 (Octoxinol 9)</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:104px;">85112</td>
			<td style="width:200px;">Detergent for permeabilization of cell membranes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Potassium hydroxide solution 1M</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">109107</td>
			<td style="width:200px;">Necessary for preparing the paraformaldehyde solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Paraformaldehyde</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">P6148</td>
			<td style="width:200px;">Fixation agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate buffered saline</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">D8537</td>
			<td style="width:200px;">Balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Chemiblocker</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">2170</td>
			<td style="width:200px;">Blocking agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Mouse monoclonal anti-&#947;H2AX antibody (JBW301)</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">05-636</td>
			<td style="width:200px;">Primary antibody for detection of &#947;H2AX</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td>
			<td style="width:119px;">Novus Biologicals</td>
			<td style="width:104px;">NB100-304</td>
			<td style="width:200px;">Primary antibody for detection of 53BP1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Alexa Fluor 488-conjugated goat anti-mouse antibody</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:104px;">A-11001</td>
			<td style="width:200px;">Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Alexa Fluor 555-conjugated goat anti-rabbit antibody</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:104px;">A-21428</td>
			<td style="width:200px;">Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Vectashield mounting medium</td>
			<td style="width:119px;">Vector Laboratories</td>
			<td style="width:104px;">H-1200</td>
			<td style="width:200px;">Contains DAPI for staining of DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Axio Scope.A1</td>
			<td style="width:119px;">Zeiss</td>
			<td style="width:104px;">490035</td>
			<td style="width:200px;">Fluorescence microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Cool Cube 1 CCD camera</td>
			<td style="width:119px;">Metasystems</td>
			<td style="width:104px;">H-0310-010-MS</td>
			<td style="width:200px;">Camera system for digital recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Isis software</td>
			<td style="width:119px;">Metasystems</td>
			<td style="width:104px;">Not applicable</td>
			<td style="width:200px;">Microscope software</td>
		</tr>
	</tbody>
</table>

immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks

DNA 二重鎖切断 (DSB) は、深刻な DNA 損傷です。DSB の修復形成の分析、ゲノム、遺伝毒性試験、放射線生物学、老化、癌、および医薬品開発を含む研究分野の広範なスペクトルに関連しています。DSB に応答して、ヒストン H2AX は蛍光顕微鏡によるセリン 139 離散核病巣検出を形成するいくつかの megabase ペアの領域内でリン酸化されます。また、53BP1 (p53 結合タンパク質 1) は別重要な DSB 応答性タンパク質相同組換えを防止しながら非相同性末端結合による DSB の修復を促進します。ΓH2AX、53BP1 の特定の機能によると γH2AX と蛍光顕微鏡により 53BP1 の複合解析 DSB の詳細な分析のための合理的なアプローチがあります。本稿では、技術を実行するための組織的なノートを添加したステップバイ ステップ プロトコルを提供します。具体的には、細胞周期の γH2AX の巣パターンに及ぼす影響は、通常細胞株培養線維芽細胞で示されます。さらに、健康な人の x 線照射によるリンパ球のマーカーとしての γH2AX の巣の値が描かれています。最後に、γH2AX、53BP1 の蛍光顕微鏡による、急性骨髄性白血病患者の CD34 陽性細胞の遺伝的不安定性を調べた。

G0/G1 期と G2 段階の場合 γH2AX 巣が (図 5 a) 異なる蛍光ドットとして表示されますセルの γH2AX 巣の分析は最も正確です。対照的に、レプリケーション プロセス (図 5 b) によって引き起こされる分散パン核 γH2AX 斑点、S 期細胞で γH2AX 巣の分析は複雑します。
細胞の固定を行った ...

Watch this Scientific Journal Video about Immunofluorescence Microscopy of ÃŽÂ³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

Immunofluorescence Microscopy of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

この原稿は、γH2AX、53BP1 の蛍光顕微鏡による DNA 二重鎖切断の分析のためのプロトコルを提供します。

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 ...

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Cancer Research

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

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

マイクロ レーザーの単一の試験と哺乳類細胞における二重鎖切断修復のためのアプリケーション

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

循環腫瘍細胞を取り込むため医療用ワイヤー線を用いた腫瘍細胞の特性解析: 蛍光抗体法や DNA 魚に基づく 3 D アプローチ

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

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

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 ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

患者の生体流体における腫瘍関連循環DNAの検出とモニタリング

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

標的抗原特異的治療抗体の腫瘍と組織分布の分析

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

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

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 ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

がん患者の血漿サンプルにおける無細胞DNAの検出

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

生物発光を用いたマウスにおける乳がん増殖および転移性コロニー形成のモニタリング

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

免疫蛍光アッセイ による 患者由来卵巣がんオルガノイド中のDNA損傷修復タンパク質の可視化(英語)

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment (Video) | JoVE 

腫瘍微小環境の臨床的および生物学的評価のための空間画像解析と組み合わせたマルチプレックス免疫蛍光法

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...