我々は、財政的に酸化ストレスの研究を支援するため、トリーゼンリヒテンシュタイン、LHW財団に感謝したいと思います。

バーゼル大学で行わコメットアッセイの研究は、眼科学科はバーゼルの現地倫理委員会によって承認されました試薬の調製<ol><li>ダルベッコのリン酸緩衝生理食塩水（PBS）（カルシウム++、Mg ++を無料）をPBS（1 Lパケット）とメディアへのdH 2 Oの990ミリリットルを追加することにより、溶液を調製します。 pHを7.4に調整します。室温で保管してください。 </li><li>溶解液を調製：1,000ミリリットルのdH 2 O中の2.5MのNaCl（146.1グラム）、100mMのEDTA（37.2グラム）および10mMのTrizma塩基（1.2グラム）を追加準備するには</li><li> 1500ミリリットルのdH 2 Oに10 M NaOHを30ミリリットルと7.5ミリリットルの0.2M EDTA、適量を添加することにより、電気泳動緩衝液（300mMのNaOHを/ 1mMのEDTA）を調製し、よく混ぜます。総容量は、ゲルボックス容量に依存します。使用前に、&gt; 13のpHを確保するために、緩衝液のpHを測定します。 </li><li> 0.4 Mトリスを含む中和バッファーを準備します（＆＃濃縮（48.5 gmは〜800ミリリットルのdH 2 Oは、7.5にpHを調整するために追加されました）62; 10 MのHCl）をdH 2 Oで千ミリリットルでこの溶液を室温で保管してください。 </li><li>染色溶液を調製します。臭化エチジウム（EtBrを、10倍の株価、20 / mlの）に室温で50ミリリットルのdH 2 Oおよび店舗でのEtBr 10mgを追加します。 1X株式の- 9ミリリットルのdH 2 Oで1ミリリットルを混ぜます慎重な！それが知られている発がん性物質であるように、適切な予防措置とのEtBrを処理します。 </li></ol>白血球の2の単離<ol><li>静脈穿刺を用いてヘパリンなどの抗凝固剤を用いたヒト血液サンプル（20ml）で取得します。 </li><li>このようなヒストとして密度勾配細胞分離器を用いてリンパ球を分離します。 <ol><li> （FBSなし）PBSまたはRPMIで1の割合と層600の上にμlの細胞分離液：簡単に言うと、1に血液を希釈。 </li><li> 4℃で20分間遠心操作し1,800×gで。 PBS / RPMI 3〜5mlのに「バフィー」のコートを吸引し、リンパ球をペレット化し、10分間300×gで遠心分離しました。 </li><li>ただPBS（RPMIとの境界上からリンパ球を取得ピペッターを使用して）および細胞分離液。プラズマと各チューブの細胞分離液層の間の界面からの白血球バンドを外し、1 50mlチューブに集めます。 </li></ol></li><li>冷ダルベッコ改変イーグル培地（DMEM）で50mlに全量を。 10分間、300×gでDMEMで細胞懸濁液を3回洗浄します。 </li><li>血球計数器を用いて、細胞の合計数をカウントします。 PBSで細胞を懸濁し、10 7細胞/チューブでマイクロ遠心チューブに等分。 </li><li>ピペットで5ミリリットル使い捨てプラスチックチューブへの細胞の0.4ミリリットル。 40℃で1.2ミリリットルの1％低ゲル化温度アガロースを追加します。混合し、迅速かつプレコートスライドのアガロースで覆われた表面上に細胞懸濁液1.2mlのをピペット。泡を生成することは避けてください。 </li><li>アガロースは、約2分間、ゲル化することができます。ゲル化のために使用される時間と温度と一致すると、そのアガロースが完全に溶解液に浸す前に設定されていることを確認。完全秒後らは、〜4ºCで溶液を溶解することに水没します。 </li></ol> 3.溶解および電気泳動注：記載された手順は、pH&gt; 13のアルカリ性条件下での電気泳動のためのものです。 <ol><li> 〜4ºCで少なくとも2時間後、静かに溶解液からスライドを削除します。場所はできるだけ一緒に近く、それらを滑り、一方の端部付近の水平ゲルボックスに並んでスライドします。 </li><li>液面が完全にスライドを覆うまで（アガロース上の泡を避けるため）焼きたてのpH&gt; 13電気泳動バッファーでバッファ貯水池を埋めます。 </li><li>スライドは、DNAとアルカリに不安定な損傷の発現の巻き戻しを可能にするために20分間、アルカリ性緩衝液に座ってみましょう。 </li><li> 25ボルト（〜0.75 V / cm）での電源をオンにして、上昇又はバッファレベルを低下させることにより、300ミリアンペアの電流を調整。 30分間スライドを電気泳動。 </li><li>電源をオフにします。静かバッファおよびpからスライドを持ち上げますドレイントレイのレース。賢明なコートを中和バッファーでスライドを削除します。スライドは、少なくとも5分間座ってすることができます。スライドを排出し、さらに2回繰り返します。 </li><li> 80μlの1×臭化エチジウム（EtBrを）でスライドを染色し、5分間放置した後、余分な汚れを除去するために冷却した蒸留水に浸し。その後、スライド上にカバースリップを置き、それらをすぐに保存します。ストアは、乾燥状態で室温で月にスライドします。 注：他のDNA染色（ 例えば 、SYBR緑）を用いてもよいです。注意！ほとんどの色素は変異原性であるように、保護手袋を着用します。 </li></ol> DNA切断の4定量<ol><li> DNA損傷を可視化するために、40倍対物レンズ蛍光顕微鏡下でのEtBr染色DNAのスライドを観察します。 </li><li> DNAの移動および移行DNA（ 図1A）の割合の長さを測定することにより定量的に細胞内のDNA損傷を評価します。 Tail-モーメントとオリーブMOMEによるDNA損傷（ 図1B）を定量化しますNT。テールモーメントとオリーブ-Momentの定義は、以前8-10を与えられています。 注：我々の経験では、それはパラメータの一つによって固執するのに十分です。細胞は、顕微鏡スライドや写真細胞上でスキャンする自動化ツールを設定することで、撮影されています。 DNA切断の定量化は、画像解析ソフトウェアパッケージによって行うことができます。カスタムセルの画像をキャプチャし、DNA損傷を定量化するために作られているいくつかの異なるソフトウェア·パッケージがあります。我々は、実験を通して同じソフトウェアパッケージを使用することをお勧めします。ラボの技術者は、無傷の細胞または彗星は、コンピューターで採点されるように識別し、その上にマウスでクリックします。彗星または無傷の細胞は、その後、自動的に採点されます。 </li></ol>

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</ol>

スウェーデンの科学者Ostlingとヨハンソンは、セル1におけるDNA損傷を定量化するためにコメットアッセイを使用して、そのフィールドの最初でした。中性条件は、二つの科学者が使用することが、唯一の二本鎖DNA切断の検出を可能にしました。シンらは 、後に二本鎖および一本鎖DNA切断を評価し、アルカリに不安定な部位12を検出することができる敏感なバージョンを生成し、アルカリ条件下で使用するためのアッセイを、適 ​​応しました。その初期の開発以来、このアッセイは、異なる細胞タイプ13-15におけるDNA損傷の種類を評価するために好適にするために様々な段階で修飾されています。 すべての技術と同様に、技術的な詳細に厳密な注意を払うことは、正確な結果16を得ることが重要です。すべての方法論的段階からの溶液製剤彗星への専門家の実験室TECHNによって実行される定量化は、されている場合は、私たちの経験に基づいて、最良の結果が得られていますicians。新鮮で正しく準備されたメディア、適切なピペッティング技術、正確なタイミングと新たに調製した白血球の（以前は結果の項で述べたように）使用の使用は、正確な結果17を得るために溶解し、ゲル電気泳動ためには不可欠です。 このアッセイのすべての個々のステップは、信頼できる結果を得るために等しく重要である。彗星定量まで溶液調製から一つ一つの方法論的なステップは、専門家の研究室によって実行された場合、一般的に最良の結果に16が得られる新鮮で正しく準備されたメディアの使用を思わtechnicians.Important 、適切なピペッティング技術、正確なタイミングとは、すでに結果セクションアッセイ17から適切な結果を得るために溶解し、ゲル電気泳動のために新たに調製した白血球の使用にいいます。 他の方法がそうであるように、コメットアッセイ技術は、アドバンがありますtagesと欠点。感度の高い方法であることは、このアッセイは、DNA切断を増強するため、結果に影響を与える可能性のある要因（ 例えば、UV光）に対して脆弱です。研究されていることを除いて、酸化的ストレスを高めることができる任意の要因は、避けるべきです。ストレッサーは、白血球ではなく、他のすべての血液媒体およびリンパ球だけでなく、酸化ストレスのレベルを上げることができます。前述したように酸化ストレス18,19の測定多くの異なる、より直接的な方法があります。コメットアッセイは、特定の利点を有している多くの方法の一つです。これらは、その比較的低いコストを含む、少数の細胞は、必要な（&lt;10,000細胞）、したがって、患者ごとに必要な少量の血液サンプル、細胞（約3日）におけるDNA損傷を定量化するのに必要な時間の比較的短い期間、その感度異なる細胞型におけるDNA損傷を査定するために、その広範な適用性。私たちはこの経験を持っていたので、過去には、我々は、白血球で動作するように選択します細胞型、それが計画された特定の研究に適用しました。コメットアッセイの別の利点は、異なるタイプおよびDNA損傷のレベルを検出するために使用することができ、そのようなDNA修復試験、補足試験または遺伝毒性試験などの酸化ストレスに加えて研究の様々な他の分野に適用することができることです。 酸化ストレスは、現在、複数の疾患の病因に重要な役割を果たしていると認識されます。コメットアッセイ法は、酸化的ストレス18,19を測定する多くの方法のうちの1つが、比較的単純な汎用性と安価です。習得した場合、この方法は、酸化ストレスが役割8-10,20,21を果たしている医学のすべての領域で使用することができます。

コメットアッセイは、最初にスウェーデンの科学者Ostlingとヨハンソン1によって開発されました。 DNAは負に荷電した分子です。電気泳動の間、小さく、損傷を受けたDNA断片は、正に荷電したアノード2に向かって速く移動します。 DNA断片が小さいほど、陽極に向かってより速くそれらの移動は、無傷の、損傷を受けていないDNAおよび損傷DNA断片3からなる「尾部」からなる「ヘッド」の典型的な「コメット」を形成します。損傷を受けたDNAは5 DNA切断の発生はかなりバランスの維持本体4内の異なるメカニズムによって修復することができます。しかし、バランスがDNA切断に賛成している場合は、ブレークは蓄積することができ、最終的に疾患の発展に貢献します。 私たちのDNAは、与えられた時間6で約0.000165パーセントのダメージを受けます。一本鎖DNAの切断は、DNA二重らせんの2本の鎖のうちの1上の欠陥を参照してください。DNA二重らせんの両方の鎖が破損しているときに、二本鎖DNA切断が発生しているのに対し。このような細胞の年齢、タバコの煙、様々な薬物や酸化ストレス7-9として与えられた時点でのDNA切断の量を高めることができる様々な要因があります。強化されたDNA切断につながるすべての要因が一定に保たれる場合には、コメットアッセイによるDNA切断の定量化は、酸化ストレスの良い間接的なパラメータです。 コメットアッセイの方法は、眼科学などの医学でますます、今日使用されています。この理由の1つは、酸化的ストレスは、より多くの種々の疾患8-11及びコメットアッセイの発症メカニズムとして認識さは酸化ストレスを間接的に定量化することが可能な1つの方法であることです。

<table><tbody><tr><td>Dimethylsulfoxide (DMSO)</td><td>Qualigens</td><td>(CPW59)</td><td></td></tr><tr><td>Disodium EDTA</td><td>HiMedia</td><td>(RM1370)</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Sigma</td><td>(E-8751)</td><td></td></tr><tr><td>Histopaque</td><td>Sigma</td><td>(1077-1)</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) (Ca++, Mg++ free)</td><td>HiMedia</td><td>(TS1006)</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Ranbaxy Rankem</td><td>(S0160)</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>BDH-Merck</td><td>(89021)</td><td></td></tr><tr><td>Triton X-100</td><td>HiMedia</td><td>(RM 845)</td><td></td></tr><tr><td>Trizma Base</td><td>Spectrochem</td><td></td><td></td></tr><tr><td colspan="4">Materials required for gel electrophoresis</td></tr><tr><td>Normal Melting Agarose (NMA)</td><td>HiMedia</td><td>(RM273)</td><td></td></tr><tr><td>Low Melting Point Agarose (LMPA)</td><td>Sigma</td><td>(A9414)</td><td></td></tr><tr><td>Methanol - Qualigens</td><td></td><td></td><td></td></tr><tr><td>Coverslips (No. 1, 24 x 60 mm)</td><td>Blue Star</td><td></td><td></td></tr><tr><td>Microcentrifuge Tubes</td><td>Tarsons</td><td>(500010)</td><td></td></tr><tr><td>Micropipettor and Tips</td><td>Tarsons</td><td></td><td></td></tr><tr><td>Microscope Slides, Conventional / Micro gel electrophoresis (MGE) slides</td><td></td><td></td><td></td></tr></tbody></table>

comet assay as an indirect measure of systemic oxidative stress

高等真核生物は酸素なしで生きることはできません。まだ、逆説的に、酸素は、それらに有害であることができます。それは別のπ*反結合軌道にある2つの不対電子を有するので、酸素分子は、化学的に比較的不活性です。これらの2つの電子が、彼らは自分自身の軸の周りに同じ方向に回転するという意味、平行スピンを持っています。酸素分子は非常に反応しないのはこのためです。酸素の活性化は、二つの異なるメカニズムによって発生することがあります。いずれかの時間（一価の減少）で一つの電子を介して還元により、または十分なエネルギーの吸収による不対電子のスピンを反転させます。これは、反応性酸化種（ROS）の産生をもたらします。人間の体は、その生理学的状態のROSを排除するいくつかの方法があります。 ROS生産は修復能力、酸化ストレスの結果と被害の異なる分子を超えた場合。酸化ストレスは私することのできる多くの異なる方法が存在しますasured。この原稿はまた、DNA切断の測定を可能にする「コメットアッセイ」として知られている細胞ゲル電気泳動、名前のメソッドのいずれかに焦点を当てています。酸化的ストレスの他のDNA損傷を引き起こすことが知られているすべての要因が一定に保たれている場合、コメットアッセイによって測定したDNA損傷の量は、酸化ストレスの良好なパラメータです。原理は単純であり、DNA分子は負に帯電しているという事実に依存します。無傷のDNA分子は、電気泳動中に移動しないような大きなサイズを有します。 DNA切断が、陽極に向かって電界内を移動より小さな断片に存在する結果であれば。小さな断片が速く移動します。フラグメントは、異なるサイズを有するように、電気泳動の最終結果は、異なるラインではなく、彗星の形状に連続ではありません。システムは、得られた「彗星」のため、細胞内のDNA切断の定量化を可能にします。

コメットアッセイの方法は、再現性のある結果を描画するが、それは様々な要因によって影響され得ます。そのような要因の1つは、白血球が溶解前、ゲル電気泳動、またはこのステップは、新たに調製した白血球の上で実行されるかどうかに凍結保存されているか否かである。 図2は 、白血球が溶解前およびゲル電気泳動SS-DNAに凍結保存したグループ2、でていることを示していますブレークは、溶解し、ゲル-electrophoresisが新たに調製された細胞で行ったグループ1に比べ、有意に高かったです。 アッセイはまた、間接的に全身酸化ストレスを評価するために使用され得ます。表1は、喫煙者と健常者でTail-モーメントとOlive-モーメントの記述統計量を示しています。データは、毎日タバコの半分パック喫煙する循環白血球9でのssDNA休憩を倍以上のことが明らかになりました。 <img alt="図1（a）" src= &quot;/ファイル/ ftp_upload / 52763 / 52763fig1A.jpg&quot; /&gt; <img alt="図1B" src="/files/ftp_upload/52763/52763fig1B.jpg" /> 図1（A）は、コメットアッセイの解析中に使用される顕微鏡を示しています。 （B）Δは明るいヘッドとテール（損傷した小さいDNA断片）を有する典型的な「彗星」を示している。 <a href="https://www.jove.com/files/ftp_upload/52763/52763fig1large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52763/52763fig2.jpg" /> 白血球は、新たに調製した白血球が溶解し、ゲル電気泳動の前に使用したときよりも（グループ2）凍結保存したときに、図2のssDNAブレークが有意に高かったです。 <imgALT = &quot;表1&quot; SRC = &quot;/ファイル/ ftp_upload / 52763 / 52763table1.jpg&quot; /&gt; 表1喫煙者は一本鎖DNAの倍以上の数が健康な非喫煙者にマッチした年齢や性別に比べて壊れていました。

Watch this Scientific Journal Video about Comet Assay as an Indirect Measure of Systemic Oxidative Stress at JoVE.com

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

コメットアッセイは、様々な要因によって誘発されるDNA破壊を測定します。 DNAの損傷を引き起こす（酸化ストレスを除く）すべての要因が一定に保持される場合、DNA損傷の量は、酸化ストレスの良い間接的なパラメータです。このプロトコルの目的は、酸化ストレスの間接的な測定のためのコメットアッセイを使用することです。

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 ...

comet-assay-as-an-indirect-measure-of-systemic-oxidative-stress

Research

JoVE Journal

Biology

29.3K ビュー.  University of Basel. コメットアッセイは、様々な要因によって誘発されるDNA破壊を測定します。 DNAの損傷を引き起こす（酸化ストレスを除く）すべての要因が一定に保持される場合、DNA損傷の量は、酸化ストレスの良い間接的なパラメータです。このプロトコルの目的は、酸化ストレスの間接的な測定のためのコメットアッセイを使用することです。 

ビデオ: Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Evaluating In Vitro DNA Damage Using Comet Assay

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 therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

コメットアッセイを用いた DNA 損傷の in Vitro評価

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 ...

がん研究

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

遺伝子にコードされた蛍光センサーを用いた毒性酸化ストレスの評価へのアプローチをイメージング

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

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

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

生物学

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

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 ...

Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum

Label the glass tubes for the samples to be analyzed, then, add 100 microliters of the prepared sample to each tube. Add 200 microliters of 8.1% SDS to each sample, and gently swirl the glass tube in a circular motion to mix.
Add 1.5 milliliters of 3.5 molar sodium acetate buffer to each sample. Then, add 1.5 milliliters of aqueous 0.8% thiobarbituric acid solution. Bring the final volume in each tube to 4 milliliters by adding 700 microliters of deionized water. Tightly cap the glass tubes, and incubate them in a heating block set to 95 degrees Celsius for 1 hour. Cover the tubes with aluminum foil to prevent condensation. Then, remove the tubes from the block and incubate them on ice for 30 minutes.
After the incubation, centrifuge the samples and standards at 1,500 x g for 10 minutes at 4 degrees Celsius. Transfer 150 microliters of supernatant from each tube to a well in a 96-well plate, removing any air bubbles with a pipette tip.
Measure the absorbance of the samples at 532 nanometers. Then, subtract the average absorbance reading of the blank samples from all other absorbance readings.
Create a standard curve and use it to calculate unknown sample concentrations.

ヒト血清中の酸化ストレスを評価するためのチオバルビツール酸反応性物質アッセイ

JoVE Encyclopedia of Experiments

生物学的手法

アッセイ技術

生化学的アッセイ

EoE Sub Articles

Comet Assay for DNA Damage Detection in Cells

To begin, prepare 0.6% low melting point agarose in PBS, and place it in a water bath at 37 degrees Celsius. Labeled the frosted end of the pre-coated slides with name, date, and treatment information using a permanent marker or pencil.
Place a chilling plate on a flat bench, and insert two frozen cooling packs into the sliding drawer below the metal surface. Then, place the slides on the chilling plate to pre-chill for 1 to 2 minutes. Centrifuge and remove the supernatant from the sample tubes, and place them immediately back on ice. Resuspend the cell pellet with 200 microliters of 0.6% low melting point agarose, and mix by pipetting.
Next, add 80 microliters of agarose-containing cells onto a chilled slide, and quickly place a coverslip on the gel. Allow the gel to set on the chilling plate for 1 to 2 minutes. In the meantime, prepare 500 milliliters of working solution of lysis buffer, and pour it into the lysis dish.
After the gels have set, remove the coverslips quickly by gently holding the slide between thumb and forefinger, and sliding the coverslip off the gel. Then, place the slides inside a slide carrier, with all the black dot marks on the slides facing in the same direction. Place the slide carrier inside the lysis dish. Close the lid of the lysis dish, and put it for incubation.
Carefully remove the slide carrier from the lysis dish without disturbing the gels. Gently, place the slide carrier in a washing dish pre-loaded with ice-cold double-distilled water. Ensure that the slides are completely submerged, and leave the setup for 30 minutes.
Place a frozen cooling pack under the electrophoresis tank to maintain optimal buffer temperature. Then, add ice-cold electrophoresis working solution to the tank, and transfer the slide carrier into it. Orient the slides, such that the cell containing gels point toward the cathode.
Incubate the slides in the electrophoresis tank for 20 minutes, to allow the DNA to unwind. Then, turn on the power supply to perform electrophoresis for 20 minutes at 1.19 volts per centimeter. After the electrophoresis is complete, turn off the power supply, and remove the slide carrier from the electrophoresis tank. Allow the slide carrier to drain on tissue paper for 30 seconds.
Next, place the slide carrier into a dish containing neutralization buffer, and leave it for 20 minutes. Following this, place it in a washing dish containing ice-cold double-distilled water, and leave it for 20 minutes. After washing, remove the slide carrier from the water, and allow the slides to dry in an incubator at 37 degrees Celsius for 1 hour.
To rehydrate the slides, transfer the slide carrier to a washing dish containing ice-cold double-distilled water and leave it for 30 minutes. Then, place the slide carrier into a staining dish containing 2.5 micrograms per milliliter propidium iodide solution.
Close the lid of the staining dish, and incubate it for 20 minutes in the dark at room temperature. After incubation, transfer the slide carrier to a separate dish, and wash it with ice-cold double-distilled water for 20 minutes.
Remove the slide carrier from the dish, and dry it completely in the dark, either in a 37 degrees Celsius incubator or at room temperature. After the slides dry completely, store them in a slide box in the dark until ready for image analysis.