この研究は、NIH、NCI、CCR の学内研究プログラムによって支えられました。すべての著者は、CCR、NCI、NIH から学内の研究助成金を受け取った。

1。 準備試薬 <ol><li>1x PBS <ol><li>900 mL dH 2 O 100 mL 10 x PBS を希釈し、pH メーターを使用して 7.4 に pH を調整します。室温で保管します</li>。
	</ol></li> <li>換散ソリューション (LS) <ol><li>2.5 M の NaCl、100 ミリメートル二ナトリウム EDTA、10 mM Tris ベース、および 200 mM NaOH 900 mL dH 2 O の準備; よく完全に溶解する混合物を許可するように約 20 分かかります。PH メーターを使用して 10 の pH を調整します。追加 1% ナトリウム ラウリル油脂肪酸サルコシンと 1% トリトン X-100 1,000 mL に最終的な音量を調整して。使用する前に、少なくとも 30 分のための 4 ° C に涼しい</li>。
	</ol></li> <li>アルカリ電気泳動ソリューション (AES)、pH &#62; 13 <ol><li>準備 200 mM NaOH と 1 mM ナトリウム EDTA 800 mL dH 2 O. 調整 pH と pH であることを確認してください &#62; 13。1,000 mL に最終的な音量を調整します。使用する前に新鮮なを確認し、使用する前に、少なくとも 30 分のための 4 ° C に冷却します</li>。
	</ol></li> <li>中立電気泳動 s sollution (ファミコン) <ol><li>氷酢酸と 100 mM 1,000 mL dH 2 O. 調整トリス酢酸ナトリウム ベースと 300 mM 9.0 pH を混合して準備 1,000 mL 中立電気泳動バッファー。使用する前に、少なくとも 30 分のための 4 ° C に涼しい</li>。
	</ol></li> <li>DNA の沈殿物の解決 (DPS) <ol><li>10 mL 7.5 M アンモニウム酢酸ストックの準備。DNA の沈殿物溶液の 50 mL 43.3 mL 95% エタノール 6.7 mL 7.5 M 酢酸アンモニウムを混在させます。室温で保管します</li>。
	</ol></li> <li>染色ソリューション <ol><li>追加 1 μ L 10,000 緑色蛍光核酸染色 (例えば、SYBR グリーン) 30 mL トリス EDTA バッファー (10 mM トリス-HCl、1 mM ナトリウム EDTA、pH 7.4) とストア x 4 ° c. の保護から光です</li>。
	</ol></li> <li>1% 低融点アガロース <ol><li>1% 低溶解点 agarose (100 mL dH 2 O で 1 g) 電子レンジで溶かします。旋回 agarose agarose が完全に溶融していることを確認するすべての 15-20 秒。使用する前に、少なくとも 20 分の 37 ° C の水浴の agarose を配置します</li>。
	</ol></li> <li>中古暖かいピペット チップ <ol><li>アガロースをピペッティングする前に 3 mm、37 ° C で暖かい P200 ピペット チップの狭い両端を切っています</li>。
	</ol></li></ol> 2。彗星のスライドを準備 <ol><li>コーティング スライド <ol><li>2 〜 3 分または agarose が完全に溶融するまで電子レンジで溶かす 1% アガロース (100 mL dH 2 O に 1 g)。Agarose にガラス顕微鏡スライドを浸し、糸くずのワイプを使用してスライドの 1 つの側面を拭く</li>。
		<li>は、空気乾燥または乾燥時間 50 ° C で熱を平らな面にスライドを置く; 乾燥後透明アガロース フィルムを形成する必要があります。使用する前に 37 ° C でコーティング スライドを配置します</li>。
	</ol></li> <li>単一細胞懸濁液の準備 <ol><li>文化と治療神経膠腫細胞 <ol><li>10 DMEM ハム F-12 培に U251 MG 細胞の培養 37 ・ # で 10 μ G/ml ストレプトマイシン、100 U/mL ペニシリン %fbs176;5% CO 2 C.</li>
			<li>は、3 分の 1 mL のトリプシンを用いた細胞を消化し、FBS と DMEM ハム F-12 培地を使用してトリプシンを中和します。収集 15 mL チューブの 4 分の 300 x g でスピン、培地を吸引、2 x 10 の 5 セル/mL の 1x PBS で細胞を中断します 。
			注: セルのサンプルは、すぐに測定を開始する前に準備されるべき、すべてのサンプルは光から DNA の損傷を防ぐために暗いまたは淡色表示環境で処理する必要があります</li>。
			<li>併用細胞懸濁液 1% 溶融低融点アガロース (37 ° C) での比率で 1:10 (v/v)、上下、ピペットで穏やかに混合し、すぐにスライドに 30 μ L をピペットします。側を使用、薄層の形成を確保する agarose/細胞の混合物を広めるためピペット チップ</li>。
			<li>10 分 30 分ゲル化時間時間を増やす高湿度環境でのサンプルの付着を向上させるため、暗闇の中で 4 ° C でフラット スライドを配置します</li>。
			<li>4 ° C LS 一晩に 1 時間の暗闇の中でスライドを没頭します</li>。
		</ol></li></ol></li></ol> 3。単一の細胞電気泳動 <ol><li>アルカリ (ステップ 3.2) に進むまたはアッセイ ニュートラル (ステップ 3.3) 彗星</li> <li>アルカリ コメットアッセイの <ol><li>そっと LS からスライドを削除、余分なバッファーをドレインし、優しく AES で 4 で 1 時間浸漬° C DNA の巻き戻しを許可します。暗闇の中のスライドを保持します</li>。
		<li>電気泳動のスライド トレイに追加済みチルド AES (電気泳動ユニットのサイズによって異なります) スライド上 0.5 cm を超えないように、内スライドとキャップ カバーを置きます。電源電圧を 1 V/cm (電極間の長さ) に設定し、4時 30 分実行 ° C</li>
		<li>ドレイン余分な電気泳動ソリューションのスライドから。DH 2 O 室温で 5 分間の 2 回のスライドをゆっくり浸す</li>。
		<li>は軽く 70% エタノール室温で 5 分間のスライドを浸します。手順 4 に進みます</li>。
	</ol></li> <li>中性コメットアッセイの <ol><li>そっと LS からスライドを削除、余分なバッファーをドレインし、4時 30 分の NES 優しく浸す ° C. 保つ暗闇の中スライド</li>。
		電気泳動スライド トレイに <li>追加中古冷蔵ニュートラル電気泳動バッファーは 0.5 cm スライド (電気泳動ユニットのサイズによって異なります) を超えていない、内側スライドとキャップ カバーを置きます。電源電圧を 1 V/cm (電極間の長さ) に設定し、4時 45 分に実行 ° C</li>
		<li>はスライドから余分なバッファーをドレイン。DPS で室温で 30 分間のスライドを軽く浸す</li>。
		<li>は軽く 70% エタノール室温で 30 分間のスライドを浸します。手順 4 に進みます</li>。
	</ol></li></ol> 4。彗星のスライドを染色 <ol><li>暗闇の中で 10-15 分の 37 ° C でスライドを乾燥します</li>。
	<li>場所 50-100 μ L 緑蛍光核酸染色液にそれぞれ乾燥アガロースと暗闇の中で室温で 15 分間染色します</li>。
	<li>DH 2 O のスライドを簡単にリンスし、暗闇の中で 37 ° C で完全に乾燥します。画像の収集及び分析に進みます</li>。
</ol> 5。画像取得・解析 注: 可視化と DNA 切断の定量化は落射蛍光顕微鏡と彗星の分析ソフトウェアに基づいています (材料の表 を参照してください) 12.
<ol><li>スライド ホルダー付け顕微鏡でスライドを配置。Agarose のゲルは対物レンズに直面していることを確認します。ランダムに 10 倍の対物レンズを用いた蛍光顕微鏡ステンド グラス彗星スライドから画像をキャプチャします。エッジと気泡の周りの領域を避ける</li>。
	<li>各彗星の尾は水平方向に分散することを確認します。彗星のヘッドが左と右から尾に属します</li>。
	<li>は、暗い背景と明るい DNA 染色バイナリ TIF 形式で各画像を保存します。ソフトウェアを使用してイメージを読み込む、&#34; 分析対象のファイルを選択して &#34; ボタンは、ツールバーの左側にあります。イメージ ビュー ウィンドウ ( 図 1) を表示する必要があります</li>。
	<li>は、画面上測定フレームを描画し、セルの彗星に従ってそのサイズを調整します。クリックして、&#34; 調整 &#34; 頭、彗星と、画像によると尾のしきい値を設定するボタンをクリック、&#34; 測定を始める &#34; ボタン ( 図 1).</li>
	<li>フレームを使用してセルを選択し、マウスでクリックして、測定をアクティブにする、、&#34; 彗星を試金 &#34; ボタン; 強度画像に現れる、&#34; プロファイル &#34; 選択した測定パラメーターを持つウィンドウ。クリックして結果を保存することができます、&#34; の結果を格納 &#34; ボタン ( 図 1). 
	注: ソフトウェアは、彗星の尾、尾の DNA、テール モーメント (TM) とオリーブ テール モーメント (OTM) の割合の長さを含むパラメーターを計算します。尾の瞬間、フォローとして数式によって計算される: <img alt="Equation 1"src="/files/ftp_upload/56450/56450eq1.jpg"/> <img alt="Equation 2"src="/files/ftp_upload/56450/56450eq2.jpg"/></li> <li>、少なくとも 50 セルを分析治療あたり</li>。
</ol>

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コメット ・ アッセイは、細胞レベルでの単一および二重鎖 DNA 切断を測定するための効率的なツールです。アッセイは、研究については遺伝毒性とバイオモニタ リング13基本病変、DNA クロスリンク、医薬品開発およびアルカリ敏感なサイトに至る「黄金の標準」として広く適用されています。本研究では、我々 はそれぞれアルカリ性と中性の彗星の試金のための 2 つの異?...

最初、1984 年は、中性条件1下核から断片を DNA の移行を示すことによっての Ostling と・ ジョハンソンによって開発コメット ・ アッセイが使用されました。テクニックは、シンら、アルカリ条件は、特異性と再現性アッセイ2に大幅に増加を示すによって後で開発されました。以来、中立コメットアッセイをほとんど使用して対し、アルカリ コメットアッセイは少量のシングルを含む、DNA 損傷に対してより敏感、二重鎖 DNA 切断、アルカリ不安定なサイト DNA DNA 二本鎖 DNA 切断を検出するか、Dna-タンパク質の架橋と不完全切除に関連付けられている DNA 一本鎖切断修復サイト3,4。両方試金断片化された DNA の可視化および DNA 損傷を定量的に評価する簡単な方法を提供できます。コメット ・ アッセイはin vitroとin vivo遺伝毒性学的研究法が機密とみなされ、初期医薬品候補の選択、環境モニタリング、人間バイオモニタ リングなど、様々 な研究分野に適用基礎 DNA 損傷研究し、5を修復します。
アッセイの原理は、電場下で断片化された DNA 核体 (「彗星頭」とも呼ばれる) のうちの移行、agarose のゲル (「彗星テール」とも呼ばれる) の DNA 汚れを形成します。ヌクレオチドは染色、この単一細胞の電気泳動によって形成された「彗星」を分析することによって DNA 損傷の程度を定量化することができます。テール モーメントの計算は、さまざまな実験的グループの間で DNA 損傷を比較するさらに助けることができます。DNA 損傷の検出の伝統的な方法と比較して、コメット ・ アッセイは直接、敏感な安価な比較的単純です。
放射線治療と化学療法は、癌治療のための一般的な戦略一本鎖の生成、二重鎖 DNA 染色体6に改します。DNA 修復阻害剤の最近の進歩の併用療法によりより効果的な遺伝毒性効果としたがって、貧血、感染症、骨髄抑制7,などの全身性の副作用が減る可能性があります。8します。 本研究で示したポリ (adp-リボース) ポリメラーゼ (PARP 阻害剤, olaparib (Ola)9の調査。PARP は豊富な核蛋白質である、10ポリ (adp-リボース) ポリマーを形成することにより DNA 塩基除去修復を担当。テモゾロミド (TMZ) は、経口投与可能なアルキル化剤で神経膠腫患者の治療に広く使用されています。DNA 損傷を定量化するコメット ・ アッセイを用いたテモゾロミドと深く olaparib を組み合わせて olaparib/テモゾロミド併用療法は神経膠腫の治療に効果的な戦略を提案する神経膠腫細胞における DNA 損傷を高めることを紹介します。テモゾロマイドだけで11と比較。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x PBS(Ca&lt;sup&gt;++&lt;/sup&gt;, Mg&lt;sup&gt;++&lt;/sup&gt; free)</td><td>TEKnova</td><td>P0196</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>EDTA</td><td>TEKnova</td><td>E0308</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>Sodium lauryl sarcosinate</td><td>Sigma</td><td>L7414</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>32318</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>695092</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma</td><td>A1542</td><td></td></tr><tr><td>SYBR Green</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Low melting point agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500</td><td></td></tr><tr><td>95% ethanol</td><td>WARNER-GRAHAM</td><td>#64-17-5</td><td></td></tr><tr><td>Trypsin</td><td>GIBICO</td><td>25300-054</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glass tissue slides</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>63422-11</td><td></td></tr><tr><td>Kimwipes</td><td>KIMberly-Clark</td><td></td><td></td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>DENVILLE</td><td></td><td></td></tr><tr><td>Pipette Tips</td><td>SHARP</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microwave</td><td>Avanti</td><td></td><td></td></tr><tr><td>Waterbath</td><td>PRECISION</td><td></td><td></td></tr><tr><td>Horizontal electrophoresis chamber</td><td>TREVIGEN</td><td>Cometassay ES II</td><td></td></tr><tr><td>Power supply</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Incubator</td><td>Quincy Lab</td><td>Model 12-140E</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Micropipettor</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

evaluating in vitro dna damage using comet assay

DNA 損傷は、その寿命の間にセルごとに一般的な現象であるし、ゲノム DNA の化学構造の変化として定義されています。ラジオや化学療法などのがんの治療法は、癌の進行を抑える細胞周期停止やアポトーシスにつながる追加の DNA 損傷の膨大な量を紹介します。実験的ガン治療中に DNA 損傷の定量評価は、遺伝毒性エージェントの有効性を正当化するための重要なステップです。本研究では単一を定量化できますコメット試験として知られている単一細胞電気泳動アッセイに着目し、体外で二重鎖 DNA の区切り。コメット ・ アッセイは、効率的かつ容易に行える、DNA 損傷定量化法、低時間/予算要求、高い再現性。ここでは、我々 は olaparib/テモゾロミド併用療法 U251 神経膠腫細胞の遺伝毒性影響を評価することで臨床研究のコメット ・ アッセイの有用性を強調表示します。

議定書は、彗星の試金実行とデータ分析 (図 1) ステップバイ ステップ ワークフローを説明します。ドキソルビシン処理 U251 細胞 (1 μ M、20 h) の彗星の尾が長く高い DNA 強度、化学療法 (図 2) により断片化された DNA のかなりの蓄積を示唆があったことを示したアルカリ性と中性の彗星の試金からの結果。
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Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Evaluating In Vitro DNA Damage Using Comet Assay

コメットアッセイを用いた DNA 損傷の in Vitro評価

コメット ・ アッセイは、単一および二重鎖 DNA を含む DNA 損傷の中断を検出する効率的な方法です。化学療法の治療効果を評価するためがん細胞における DNA 損傷を測定するアルカリ性と中性の彗星の試金を記述します。

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

evaluating-in-vitro-dna-damage-using-comet-assay

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay

37.0K ビュー.  National Cancer Institute. コメット ・ アッセイは、単一および二重鎖 DNA を含む DNA 損傷の中断を検出する効率的な方法です。化学療法の治療効果を評価するためがん細胞における DNA 損傷を測定するアルカリ性と中性の彗星の試金を記述します。

ビデオ: Evaluating In Vitro DNA Damage Using Comet Assay

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

がん研究

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

発芽アッセイ回転楕円体を使用して推定される芽新生体外パフォーマンスを評価する包括的な手順

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip：マイクロアレイヒト細胞におけるDNA損傷を測定するためのハイスループット96ウェルプラットフォーム

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

生物工学

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

DNA損傷評価のための彗星アッセイにおける凍結組織の利用

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

遺伝学

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

コメットアッセイを用いて海馬神経細胞のDNA損傷を測定する

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

神経科学

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

全身酸化ストレスの間接的な指標として、コメットアッセイ

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

生物学

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

ラットの肝臓におけるDNA鎖切断および酸化的DNA損傷を測定するためのインビボアルカリコメットアッセイおよび酵素修飾されたアルカリコメットアッセイで

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

細胞DNA損傷を評価するためのハイスループットコメットアッセイアプローチ

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy

Source: Lu, Y., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56450">Evaluating In Vitro DNA Damage Using Comet Assay</a> J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.

アルカリ性単一細胞ゲル電気泳動:化学療法後の個々の癌細胞におけるDNA損傷の定量的推定のための高感度技術

Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell ...

JoVE Encyclopedia of Experiments

生物学的手法

電気泳動技術

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

細胞内のDNA損傷検出のためのCometアッセイ

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...