Name: comet assay as an indirect measure of systemic oxidative stress
Uploaded: 2015-05-22T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Comet Assay as an Indirect Measure of Systemic Oxidative Stress at JoVE.com

Vorremmo ringraziare il ERP Stiftung, in Triesen Liechtenstein, per sostenere finanziariamente la nostra ricerca sullo stress ossidativo.

Studi sul test della cometa eseguite presso l&#39;Università di Basilea, Dipartimento di Oftalmologia sono stati approvati dal Comitato Etico Locale di Basilea 1. Preparazione dei reagenti <ol><li> Preparare Dulbecco tampone fosfato (PBS) (Ca ++, Mg ++ libero) soluzione aggiungendo PBS (1 L confezione) e 990 ml di dH 2 O ai media. Regolare il pH a 7,4. Conservare a temperatura ambiente. </li><li> Preparare la soluzione di lisi: Per preparare 1.000 ml aggiungono 2,5 M NaCl (146,1 g), EDTA 100 mm (37,2 g) e 10 Trizma mm base (1,2 g) in dH 2 O. </li><li> Preparare buffer per elettroforesi (NaOH 300 mm / 1 mM EDTA) con l&#39;aggiunta di 30 ml di NaOH 10 M e 7,5 ml di 0,2 M EDTA, qb a 1500 ml dH 2 O e mescolare bene. Il volume totale dipende dalla capacità di dialogo gel. Prima dell&#39;uso, misurare il pH del tampone per assicurare un pH&gt; 13. </li><li> Preparare tampone neutralizzazione contenente 0,4 M Tris (48.5 gm aggiunto a ~ 800 ml dH 2 O, aggiustare il pH a 7,5), concentrato (&amp; #62; 10 M HCl) a 1000 ml con dH 2 O. Conservare la soluzione a temperatura ambiente. </li><li> Preparare soluzione colorante. Per etidio bromuro (EtBr, 10x Archivio, 20 mg / ml) aggiungere 10 mg di EtBr in 50 ml di dH 2 O e conservare a temperatura ambiente. Per 1x Archivio - mescolare 1 ml con 9 ml dH 2 O. PRUDENTE! Maneggiare EtBr con adeguata cautela in quanto è un noto cancerogeno. </li></ol> 2. Isolamento dei leucociti <ol><li> Ottenere campioni umani di sangue (20 ml) con anticoagulante come l&#39;eparina mediante puntura venosa. </li><li> Separare i linfociti utilizzando un separatore cellulare gradiente di densità come Histopaque. <ol><li> Brevemente, diluire il sangue in rapporto 1: 1 con PBS o RPMI (senza FBS) e lo strato di oltre 600 microlitri separatore cellulare liquido. </li><li> Centrifugare 1.800 xg per 20 min a 4 ° C. Aspirare il coat &#39;buffy&#39; in 3-5 ml di PBS / RPMI e centrifugato a 300 xg per 10 minuti per ottenere il pellet linfociti. </li><li> Recuperare i linfociti da appena sopra confine tra PBS (RPMI) E separatore cellulare liquido, con una pipetta. Rimuovere le bande leucociti dall&#39;interfaccia tra il plasma e il separatore di cella strati liquidi di ciascun tubo e raccogliere in una provetta da 50 ml. </li></ol></li><li> Portare il volume totale di 50 ml con il freddo di Dulbecco modificato (DMEM). Lavare la sospensione cellulare tre volte con DMEM a 300 xg per 10 min. </li><li> Contare il numero totale di cellule utilizzando un emocitometro. Sospendere le cellule in PBS e aliquote in provette da microcentrifuga a 10 7 cellule / tubo. </li><li> Pipettare 0,4 ml di cellule in 5 ml di plastica tubo usa e getta. Aggiungere 1,2 ml di 1% a bassa temperatura di gelificazione agarosio a 40 ° C. Mescolare e rapidamente e pipettare 1,2 ml di sospensione cellulare sulla superficie agarosio-coperto di un vetrino pre-rivestite. Evitare la produzione di bollicine. </li><li> Lasciare che il agarosio di gel per circa 2 minuti. Essere coerenti con il tempo e la temperatura utilizzata per la gelificazione, e garantire che agarosio sia completamente impostata prima di immergere in soluzione di lisi. Dopo completamente set, immergere in soluzione lisante a ~ 4 ° C. </li></ol> 3. Lysis e elettroforesi NOTA: La procedura descritta è per elettroforesi in condizioni di pH&gt; 13 alcaline. <ol><li> Dopo almeno 2 ore a ~ 4 ° C, rimuovere delicatamente i vetrini dalla soluzione di lisi. Collocare i vetrini affiancati sulla scatola gel orizzontale vicino ad una estremità, scorrevole come vicino possibile. </li><li> Riempire i serbatoi tampone con appena fatto tampone pH&gt; 13 elettroforesi fino a quando il livello del liquido copra completamente le diapositive (evitare bolle sul agarosio). </li><li> Lasciare diapositive sedersi in tampone alcalino per 20 min per consentire lo svolgimento del DNA e l&#39;espressione di danni alcali-labili. </li><li> Attivare alimentazione a 25 volt (~ 0,75 V / cm) e regolare la corrente a 300 milliampere alzando o abbassando il livello del buffer. Elettroforesi i vetrini per 30 min. </li><li> Spegnere l&#39;alimentazione. Sollevare delicatamente le diapositive dal buffer e ppizzo su un vassoio di scarico. Eliminare cappotto saggio i vetrini con la neutralizzazione Buffer. Lasciare i vetrini a sedere per almeno 5 minuti. Scolare diapositive e ripetere altre due volte. </li><li> Colorare i vetrini con 80 microlitri 1x etidio bromuro (EtBr) e lasciar riposare per 5 minuti e poi immergere in acqua distillata fredda per eliminare l&#39;eccesso macchia. Poi posto un vetrino sul vetrino e archiviare immediatamente. Conservare i vetrini per un mese a temperatura ambiente in condizioni di asciutto. NOTA: Altri macchie DNA (ad esempio, SYBR-verde) può essere utilizzato anche. Attenzione! indossare guanti di protezione, come la maggior parte delle tinture sono mutageni. </li></ol> 4. Quantificazione delle rotture del DNA <ol><li> Per visualizzare il danno al DNA, di osservare il vetrino DNA EtBr macchiato con un microscopio a fluorescenza obiettivo 40X. </li><li> Valutare danni DNA quantitativamente nelle cellule misurando la lunghezza di migrazione del DNA e la percentuale di DNA migrato (Figura 1A). Quantificare il danno al DNA (Figura 1B) dal momento in cui parti apribili e Olive-Moment. Definizioni di Tail-Moment e Olive -Moment sono stati precedentemente data 8-10. NOTA: Nella nostra esperienza è abbastanza per attaccare da uno dei parametri. Le cellule sono fotografati impostando lo strumento automatico che analizza sulle celle vetrino da microscopio e fotografie. Quantificazione delle rotture del DNA può essere effettuata da pacchetti software di analisi delle immagini. Ci sono diversi pacchetti software differenti che sono realizzati su misura per catturare l&#39;immagine delle cellule e quantificare il danno al DNA. Si consiglia di utilizzare lo stesso pacchetto software tutto l&#39;esperimento. Il tecnico di laboratorio identifica la cellula intatta o una cometa da valutare sul computer e fa clic con il mouse su di esso. La cella cometa o intatto viene segnato automaticamente. </li></ol>

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Scienziati svedesi Ostling e Johanson sono stati i primi nel loro campo di utilizzare test della cometa per quantificare i danni del DNA nelle cellule 1. Le condizioni neutre che i due scienziati hanno usato, tuttavia, solo permesso la rilevazione di doppio filamento rotture del DNA. Singh et al. In seguito adattato il test per l&#39;uso in ambiente alcalino, che ha prodotto una versione sensibile che potrebbe valutare doppie e singole filamento rotture del DNA e di rilevare i siti alcali-labili 12. Dal suo sviluppo iniziale, il test è stato modificato in varie fasi per renderlo adatto per valutare i tipi di danno al DNA in diversi tipi cellulari 13-15. Come per tutte le tecniche, prestando una rigorosa attenzione ai dettagli tecnici è importante per ottenere risultati accurati 16. Sulla base della nostra esperienza, i migliori risultati si ottengono se ogni passo da metodologico preparazione soluzione cometa quantificazione-viene eseguita da esperti tecn laboratorioicians. L&#39;utilizzo di mezzi freschi e correttamente preparato, adeguate tecniche di pipettatura, esatta tempistica e (come precedentemente menzionato nella sezione risultati) l&#39;uso di leucociti preparati al momento sono essenziali per lisi e elettroforesi su gel di ottenere risultati accurati 17. Tutte le singole fasi di questo saggio sono ugualmente importanti per ottenere risultati affidabili. 16 In generale i migliori risultati si ottengono se ogni singolo passo metodologico dalla preparazione soluzione fino quantificazione cometa viene eseguita da esperti del laboratorio technicians.Important sembrano l&#39;uso di mezzi freschi e correttamente preparati , adeguate tecniche di pipettatura, i tempi esatti e, come già di cui nella sezione dei risultati l&#39;uso di leucociti preparati al momento per lisi e gel elettroforesi per ottenere risultati adeguati dal saggio 17. Come fare altre metodologie, la tecnica comet assay ha il suo advanTages e svantaggi. Essendo un metodo sensibile, il saggio è vulnerabile a fattori (ad esempio, raggi UV) che possono aumentare la rotture del DNA e quindi influenzare i risultati. Qualsiasi fattore che può aumentare lo stress ossidativo, ad eccezione di ciò che è oggetto di ricerca, dovrebbe essere evitato. Stress possono aumentare il livello di stress ossidativo non solo nei leucociti, ma anche in tutti gli altri mezzi di sangue e linfociti. Come detto in precedenza ci sono molti modi diversi e più dirette di misurare lo stress ossidativo 18,19. Il saggio comet è uno dei molti metodi che ha alcuni vantaggi. Questi includono il suo costo relativamente basso, il piccolo numero di celle richieste (&lt;10.000 cellule) e quindi i piccoli campioni di sangue necessaria per paziente, il periodo relativamente breve di tempo necessario per quantificare il danno del DNA in cellule (circa 3 giorni), la sua sensibilità e la sua diffusa applicabilità ai danni asini DNA in diversi tipi di cellule. In passato abbiamo scegliere di lavorare con leucociti da quando abbiamo avuto esperienza con questotipo cellulare ed era applicabile per un particolare studio previsto. Un altro vantaggio del comet assay è che può essere utilizzato per rilevare diversi tipi e livelli di danno al DNA e quindi può essere applicata in vari altri settori di studi oltre stress ossidativo quali studi di riparazione del DNA, trattamenti per il supporto o genotossicità. Lo stress ossidativo è ora riconosciuto come giocare un ruolo fondamentale nella patogenesi di diverse malattie. Il metodo comet assay, mentre uno dei molti modi per misurare lo stress ossidativo 18,19, è relativamente semplice, versatile e poco costoso. Quando masterizzato, questo metodo può essere utilizzato in tutti i settori della medicina in cui lo stress ossidativo svolge un ruolo significativo 8-10,20,21.

Comet assay è stato sviluppato da scienziati svedesi Ostling e Johanson 1. DNA è una molecola carica negativa. Durante l&#39;elettroforesi, più piccole, i frammenti di DNA danneggiato viaggiano più velocemente verso un anodo caricato positivamente 2. Più i frammenti di DNA, più veloce la loro migrazione verso l&#39;anodo a formare un tipico &quot;comet&quot; con una &quot;testa&quot; composta intatta, DNA integro e una &quot;coda&quot; composto di frammenti di DNA danneggiati 3. DNA danneggiato può essere riparato da diversi meccanismi nel corpo 4, che mantengono la generazione di rotture del DNA abbastanza equilibrati 5. Se, tuttavia, il saldo è a favore di rotture del DNA, le interruzioni possono accumularsi e finiranno contribuire allo sviluppo di una malattia. Il nostro DNA subisce circa 0.000165% di danni in un determinato momento 6. Singole rotture del DNA bloccati si riferiscono ad un difetto su uno dei due filamenti del DNA a doppia elicaconsiderando double-stranded rotture del DNA sono causati quando entrambi i filamenti della doppia elica del DNA sono danneggiati. Ci sono vari fattori che possono aumentare la quantità di rotture del DNA in un dato momento, quali l&#39;età della cellula, fumo di sigaretta, vari farmaci o stress ossidativo 7-9. Se tutti i fattori che portano a rotture del DNA avanzati sono mantenute costanti, quindi la quantificazione di rotture del DNA per mezzo di test della cometa è un buon parametro indiretto dello stress ossidativo. Il metodo di test della cometa è utilizzata sempre più oggi in medicina tra cui oftalmologia. Una ragione di ciò è che lo stress ossidativo è sempre più riconosciuto come un meccanismo patogenetico per varie malattie 8-11 e comet assay è un metodo con cui lo stress ossidativo può indirettamente essere quantificato.

<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

Organismi eucarioti superiori non possono vivere senza ossigeno; tuttavia, paradossalmente, l&#39;ossigeno può essere dannoso per loro. La molecola di ossigeno è chimicamente inerte, relativamente perché ha due elettroni spaiati situati in diverse pi * orbitali anti-incollaggio. Questi due elettroni hanno spin paralleli, nel senso che ruotano nello stesso verso attorno al proprio asse. Per questo motivo la molecola di ossigeno non è molto reattivo. Attivazione di ossigeno può avvenire per due meccanismi differenti; sia attraverso la riduzione con un elettrone alla volta (riduzione monovalente), oppure attraverso l&#39;assorbimento di energia sufficiente per invertire la rotazione di uno degli elettroni spaiati. Il risultato è la produzione di specie reattive ossidative (ROS). Ci sono un certo numero di modi in cui il corpo umano elimina ROS nel suo stato fisiologico. Se la produzione di ROS supera la capacità di riparazione, i risultati dello stress ossidativo e danni molecole diverse. Ci sono molti metodi diversi con cui lo stress ossidativo può essere measured. Questo manoscritto concentra su uno dei metodi denominati gel elettroforesi cellulare, noto anche come &quot;comet assay&quot;, che consente la misurazione di rotture del DNA. Se tutti i fattori che causano danni al DNA, diverso da stress ossidativo sono mantenute costanti, la quantità di danno al DNA determinato tramite test cometa è un buon parametro di stress ossidativo. Il principio è semplice e si basa sul fatto che le molecole di DNA sono caricate negativamente. Una molecola di DNA intatto ha una grande dimensione tale da non migra durante l&#39;elettroforesi. Rotture del DNA, tuttavia, se presente risultato in frammenti più piccoli che si muovono nel campo elettrico verso l&#39;anodo. Frammenti più piccoli migrano più velocemente. Poiché i frammenti hanno dimensioni diverse il risultato finale della elettroforesi non è una linea distinta ma piuttosto un continuum con la forma di una cometa. Il sistema consente di quantificare il &quot;comet&quot; risultante e quindi delle rotture del DNA nella cellula.

Sebbene il metodo di test della cometa rende risultati riproducibili, può essere influenzata da una varietà di fattori. Uno di questi fattori è o meno i leucociti vengono crioconservati prima lisi e gel-elettroforesi o se questa operazione viene eseguita su leucociti appena preparati. La Figura 2 mostra che nel gruppo 2, dove sono stati crioconservati prima lisi e l&#39;elettroforesi su gel ss- DNA leucociti pause erano significativamente più alti, rispetto al gruppo 1 dove lisi e gel -Elettroforesi è stato fatto su cellule preparate al momento. Il dosaggio può anche essere utilizzato per valutare indirettamente stress ossidativo sistemico. La tabella 1 mostra le statistiche descrittive per fanalino posteriore momento e Olive- momento in fumatori e soggetti sani. I dati rivelano che il fumo mezzo pacchetto di sigarette al giorno raddoppia pause ssDNA nei leucociti circolanti 9. <img alt="Figura 1A" src= &quot;/ Files / ftp_upload / 52763 / 52763fig1A.jpg&quot; /&gt; <img alt="Figura 1B" src="/files/ftp_upload/52763/52763fig1B.jpg" /> Figura 1. (A) raffigura il microscopio utilizzato durante l&#39;analisi del test della cometa. (B) Δ raffigura una tipica &quot;cometa&quot; con una testa brillante e la coda (danneggiato frammenti di DNA più piccoli). <a href="https://www.jove.com/files/ftp_upload/52763/52763fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52763/52763fig2.jpg" /> Figura 2. pause ssDNA erano significativamente più alti quando i leucociti sono stati crioconservati (Gruppo 2) rispetto a quando leucociti preparati al momento sono stati utilizzati prima di lisi e gel elettroforesi. <imgalt = &quot;tabella 1&quot; src = &quot;/ files / ftp_upload / 52763 / 52763table1.jpg&quot; /&gt; Tabella 1. I fumatori avevano più del doppio del numero di interruzioni ssDNA rispetto a età e sesso abbinato non fumatori sani.

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

Comet assay misura rotture del DNA, indotte da fattori diversi. Se tutti i fattori (ad eccezione di stress ossidativo) che causano danni al DNA sono mantenute costanti, la quantità di danni al DNA è un buon parametro indiretto dello stress ossidativo. L&#39;obiettivo di questo protocollo è quello di utilizzare comet assay per la misura indiretta di stress ossidativo.

Comet Assay come una misura indiretta della sistemica Stress ossidativo

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

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29.3K Views.  University of Basel.  Comet assay misura rotture del DNA, indotte da fattori diversi. Se tutti i fattori (ad eccezione di stress ossidativo) che causano danni al DNA sono mantenute costanti, la quantità di danni al DNA è un buon parametro indiretto dello stress ossidativo. L'obiettivo di questo protocollo è quello di utilizzare comet assay per la misura indiretta di stress ossidativo. 

Video: 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.

Valutazione In Vitro del DNA danni utilizzando test della cometa

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 ...

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Ricerca sul cancro

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.

Approcci alla valutazione dello Stress ossidativo tossicologici utilizzando geneticamente codificato Fluorogenic sensori di imaging

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: A High-throughput di 96-Well Piattaforma per la misurazione dei danni cagionati DNA in cellule umane Microarrayed

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

Bioingegneria

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.

Un test per misurare capacità di risposta comportamentale di Zebrafish a variazioni di intensità di luce

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 ...

Biologia

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.

Uso di tessuto congelato nel saggio Cometa per la valutazione del danno del DNA

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 ...

Genetica

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.

Il saggio danneggiare il DNA in neuroni ippocampali Utilizzo del 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 ...

Neuroscienze

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.

In Vivo alcalina Comet Assay e l&#39;enzima modificato alcalina Comet Assay per la misurazione rotture del DNA Strand e danno ossidativo del DNA in fegato di ratto

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.

Un approccio di analisi delle comete ad alto rendimento per la valutazione del danno al DNA cellulare

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.

Dosaggio delle sostanze reattive all'acido tiobarbiturico per valutare lo stress ossidativo nel siero umano

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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.

Comet Assay come una misura indiretta della sistemica Stress ossidativo

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