Questa ricerca è stata sostenuta dal programma di ricerca intramurale del NIH, NCI e CCR. Tutti gli autori ricevevano assegno di ricerca intramurale da NIH, NCI e CCR.

1. preparare i reagenti <ol><li>1X PBS <ol><li>diluire 100 mL di PBS 10X con 900 mL dH 2 O e regolare il pH a 7,4 utilizzando un pH-metro. Conservare a temperatura ambiente.</li>
	</ol></li> <li>Soluzione di lisi (LS) <ol><li>preparare 2,5 M NaCl, EDTA disodico 100 mM, 10 mM Tris base e 200mm NaOH in 900 mL dH 2 O; prende comunemente circa 20 min per consentire la miscela dissolvere completamente. Regolare il pH a 10 utilizzando un pH-metro. Aggiungere 1% sodio lauryl sarcosinate e 1% Triton X-100 e regolare il volume finale a 1.000 mL. Raffreddare a 4 ° C per almeno 30 min prima dell'uso.</li>
	</ol></li> <li>Soluzione l'elettroforesi alcalina (AES), pH &#62; 13 <ol><li>preparare 200mm NaOH e 1 mM EDTA disodico in 800 mL dH 2 O. regolare il pH e assicurarsi che è pH &#62; 13. Regolare il volume finale a 1.000 mL. Fare fresco prima dell'uso e raffreddare a 4 ° C per almeno 30 min prima dell'uso.</li>
	</ol></li> <li>Neutro elettroforesi sollution (NES) <ol><li>preparare 1.000 mL di tampone di elettroforesi neutro miscelando 100 mM Tris base e 300 mM sodio acetato a 1.000 mL dH 2 O. regolare il pH a 9.0 con acido acetico glaciale. Raffreddare a 4 ° C per almeno 30 min prima dell'uso.</li>
	</ol></li> <li>Soluzione di precipitazione del DNA (DPS) <ol><li>preparazione di 10 mL brodo di 7.5 M ammonio acetato. Per 50 mL di soluzione di precipitazione del DNA, mescolare 6,7 mL 7,5 M di acetato di ammonio con 43,3 mL di etanolo al 95%. Conservare a temperatura ambiente.</li>
	</ol></li> <li>Soluzione di colorazione <ol><li>aggiungere 1 µ l 10.000 x macchia verde fluorescente dell'acido nucleico (ad es., SYBR Green) in tampone di 30 mL di Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA bisodico, pH 7,4) e conservare a 4 ° C. Protect da luce.</li>
	</ol></li> <li>1% bassa fusione dell'agarosi <ol><li>sciogliere 1% bassa fusione punto agarosio (1 g in 100 mL dH 2 O) in un forno a microonde. Agitare l'agarosio ogni 15-20 s per assicurarsi che l'agarosio è completamente fusa. Posizionare l'agarosio in bagnomaria a 37 ° C per almeno 20 minuti prima dell'uso.</li>
	</ol></li> <li>Pre-riscaldare puntali per <ol><li>tagliato le estremità strette di puntali per pipette P200 da 3 mm e calda a 37 ° C prima di pipettaggio agarosio.</li>
	</ol></li></ol> 2. Preparare la cometa scivola <ol><li>Slide rivestimento <ol><li>Melt agarosio all'1% (1 g in 100 mL dH 2 O) in un forno a microonde per 2-3 minuti o fino a quando l'agarosio è completamente fusa. Immergere i vetrini in agarosio e strofinare un lato della diapositiva servendosi di un panno privo di lanugine.</li>
		<li>Disporre i vetrini su una superficie piana per asciugare all'aria o scaldare a 50 ° C per asciugatura più rapida; un film trasparente dell'agarosi dovrebbe essere formato dopo l'essiccazione. Porre i vetrini rivestiti a 37 ° C prima dell'uso.</li>
	</ol></li> <li>Preparazione delle sospensioni di cellule singole <ol><li>cultura e trattare le cellule di glioma <ol><li>cultura le cellule U251 MG in medium DMEM-Ham F-12 supplementato con 10% FBS, 100 U/mL di penicillina e 10 µ g/mL di streptomicina a 37 & n. 176; C con 5% CO 2.</li>
			<li>Digerire le cellule con tripsina 1 mL per 3 min e neutralizzare la tripsina usando mezzo DMEM-Ham F-12 con FBS. Raccogliere in provetta da 15 mL, girare a 300 x g per 4 min, aspirare il mezzo e sospendere le cellule a 2 x 10 5 cellule/mL in PBS 1X. 
			Nota: Il campione di cellule dovrebbe essere preparato immediatamente prima di iniziare l'analisi e tutti i campioni devono essere manipolati in un ambiente scuro o grigio per impedire il danno del DNA dalla luce.</li>
			<li>Unire la sospensione cellulare con agarosio all'1% fuso basso punto di fusione (a 37 ° C) con un rapporto 01:10 (v/v), mescolare delicatamente pipettando su e giù e immediatamente una pipetta 30 µ l in una diapositiva. Usare il lato della la punta della pipetta di diffondere la miscela di agarosio/cella per garantire la formazione di un sottile strato.</li>
			<li>Mettere il vetrino piatta a 4 ° C al buio per 10 minuti, aumentando il tempo di gelificazione a 30 min migliora l'aderenza dei campioni in ambienti con elevata umidità.</li>
			<li>Immergere il vetrino in 4 ° C LS al buio per 1 h a tutta la notte.</li>
		</ol></li></ol></li></ol> 3. Singola cella elettroforesi <ol><li>Vai al alcalina (passaggio 3.2) o neutro (punto 3.3) cometa dosaggio</li> <li>per analisi alcalina della cometa <ol><li>delicatamente togliere i vetrini dalla LS, scarico tampone in eccesso e immergere delicatamente in AES per 1 h a 4 ° C per consentire la rimozione del DNA. Mantenere le diapositive al buio.</li>
		<li>Aggiungi AES pre-refrigerati nel vassoio scorrevole elettroforesi, non superare 0,5 cm di sopra le diapositive (questo dipende dalle dimensioni delle unità elettroforesi), posizionare le diapositive all'interno e coprire con un coperchio. Impostare la tensione di alimentazione a 1 V/cm (la lunghezza tra gli elettrodi) ed eseguire per 30 min a 4 ° C.</li>
		<li>Soluzione di elettroforesi in eccesso di scarico da diapositiva. Immergere delicatamente i vetrini due volte in dH 2 O per 5 minuti ciascuno, a temperatura ambiente.</li>
		<li>Delicatamente immergere i vetrini in etanolo al 70% per 5 min a temperatura ambiente. Procedere al passaggio 4.</li>
	</ol></li> <li>Per analisi neutrale della cometa <ol><li>rimuovere delicatamente i vetrini dalla LS, scarico tampone in eccesso e immergere delicatamente a NES per 30 min a 4 ° C. Keep la diapositiva al buio.</li>
		Buffer di pre-refrigerati elettroforesi neutro <li>Aggiungi nella barra delle applicazioni di elettroforesi diapositiva, non superare 0,5 cm sopra diapositive (questo dipende dalle dimensioni delle unità elettroforesi), posizionare le diapositive all'interno e coprire con un coperchio. Impostare la tensione di alimentazione a 1 V/cm (la lunghezza tra gli elettrodi) ed eseguire per 45 min a 4 ° C.</li>
		<li>Scarico tampone in eccesso dalle diapositive. Immergere delicatamente i vetrini in DPS per 30 min a temperatura ambiente.</li>
		<li>Delicatamente immergere i vetrini in etanolo al 70% per 30 min a temperatura ambiente. Procedere al passaggio 4.</li>
	</ol></li></ol> 4. Colorare vetrini cometa <ol><li>a secco diapositive a 37 ° C per 10-15 min al buio.</li>
	<li>Posto 50-100 µ l verde fluorescente dell'acido nucleico macchiatura soluzione su ogni secchi agarosio e macchia per 15 min a temperatura ambiente al buio.</li>
	<li>Sciacquare brevemente le diapositive in dH 2 O e asciugare completamente a 37 ° C al buio. Procedere all'acquisizione di immagini e analisi.</li>
</ol> 5. Acquisizione e analisi di immagine Nota: la visualizzazione e la quantificazione delle rotture del DNA si basano su epifluorescenza e il software di dosaggio di cometa (Vedi Tabella materiali) 12 .
<ol><li>Porre i vetrini al microscopio con un supporto per diapositive. Accertarsi che il gel dell'agarosi è rivolta alla lente dell'obiettivo. In modo casuale e catturare immagini dalle diapositive cometa macchiato con un microscopio a fluorescenza con una lente di obiettivo 10x. Evitare i bordi e le zone intorno a bolle d'aria.</li>
	<li>Assicurarsi ogni coda di cometa è distribuito in orizzontale. Teste di cometa devono provenire da sinistra e la coda da destra.</li>
	<li>Salvare ogni immagine in un formato binario TIF con brillante macchia di DNA e sfondo scuro. Caricare immagini per il software utilizzando il &#34; selezionare i file da analizzare &#34; pulsante, che si trova sulla sinistra della barra degli strumenti. Una finestra di visualizzazione immagine dovrebbe apparire ( Figura 1).</li>
	<li>Disegnare un quadro di misura sullo schermo e regolarne le dimensioni secondo la cometa della cella. Clicca la &#34; regolare &#34; pulsante per impostare la soglia della testa, cometa e coda secondo l'immagine, quindi fare clic sui &#34; iniziare la misurazione &#34; pulsante ( Figura 1).</li>
	<li>Selezionare una cella utilizzando il telaio e attivare la misura cliccando con il mouse sul &#34; la cometa di analisi &#34; pulsante; un'intensità immagine si presenta sul &#34; profili &#34; finestra con i parametri di misura selezionata. I risultati possono essere salvati cliccando il &#34; memorizzare il risultato &#34; pulsante ( Figura 1). 
	Nota: Il software calcola i parametri tra cui la lunghezza della coda di cometa, la percentuale di DNA dalla coda, il momento di coda (TM) e il momento di coda d'oliva (OTM). I momenti di coda sono calcolati mediante le formule come segue: <img alt="Equation 1" src="/files/ftp_upload/56450/56450eq1.jpg" /> <img alt="Equation 2" src="/files/ftp_upload/56450/56450eq2.jpg" /></li> <li>analizzare almeno 50 cellule per ogni trattamento.</li>
</ol>

<ol>
	<li>Ostling, O., Johanson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochem Biophys Res Commun. 123 (1), 291-298 (1984).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells.">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp Cell Res. 175 (1), 184-191 (1988).</li><li>Tice, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+gel/comet+assay:+guidelines+for+in+vitro+and+in+vivo+genetic+toxicology+testing.">Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.</a> Environ Mol Mutagen. 35 (3), 206-221 (2000).</li><li>Shah, A. J., Lakkad, B. C., Rao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+in+lead+treated+human+lymphocytes+evaluated+by+micronucleus+and+comet+assays.">Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.</a> Indian J Exp Biol. 54 (8), 502-508 (2016).</li><li>Azqueta, A., Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch Toxicol. 87 (6), 949-968 (2013).</li><li>Goldstein, M., Kastan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+implications+for+tumor+responses+to+radiation+and+chemotherapy.">The DNA damage response: implications for tumor responses to radiation and chemotherapy.</a> Annu Rev Med. 66, 129-143 (2015).</li><li>Gavande, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+targeted+therapy:+The+past+or+future+of+cancer+treatment?.">DNA repair targeted therapy: The past or future of cancer treatment?.</a> Pharmacol Ther. 160, 65-83 (2016).</li><li>Torgovnick, A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+mechanisms+in+cancer+development+and+therapy.">DNA repair mechanisms in cancer development and therapy.</a> Front Genet. 6, 157 (2015).</li><li>Weston, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARP+inhibitor+olaparib+induces+significant+killing+of+ATM-deficient+lymphoid+tumor+cells+in+vitro+and+in+vivo.">The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.</a> Blood. 116 (22), 4578-4587 (2010).</li><li>Brown, J. S., O'Carrigan, B., Jackson, S. P., Yap, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+Repair+in+Cancer:+Beyond+PARP+Inhibitors.">Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.</a> Cancer Discov. 7 (1), 20-37 (2017).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensitivity+of+IDH1-Mutated+Gliomas+Due+to+an+Impairment+in+PARP1-Mediated+DNA+Repair.">Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.</a> Cancer Res. 77 (7), 1709-1718 (2017).</li><li>Konca, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cross-platform+public+domain+PC+image-analysis+program+for+the+comet+assay.">A cross-platform public domain PC image-analysis program for the comet assay.</a> Mutat Res. 534 (1-2), 15-20 (2003).</li><li>Valverde, M., Rojas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+occupational+biomonitoring+using+the+Comet+assay.">Environmental and occupational biomonitoring using the Comet assay.</a> Mutat Res. 681 (1), 93-109 (2009).</li><li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+for+DNA+damage+and+repair:+principles,+applications,+and+limitations.">The comet assay for DNA damage and repair: principles, applications, and limitations.</a> Mol Biotechnol. 26 (3), 249-261 (2004).</li><li>Karbaschi, M., Cooke, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+for+the+high-throughput+processing+of+slides+for+the+comet+assay.">Novel method for the high-throughput processing of slides for the comet assay.</a> Sci Rep. 4, 7200 (2014).</li></ol>

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Il test della cometa è uno strumento efficace per misurare le rotture di singolo e doppio filamento del DNA a livello cellulare. Il dosaggio è stato ampiamente applicato come un "golden standard" negli studi per quanto riguarda la genotossicità e biomonitoraggio13, che vanno dalle lesioni della base, legami incrociati del DNA, lo sviluppo di farmaci e siti sensibili dell'alcali. Nello studio presente, abbiamo mostrato due distinti protocolli passo-passo per i dosaggi cometa alcalino e neutro, r...

Il test della cometa è stato sviluppato da Ostling e Johanson nel 1984 dimostrando che la migrazione del DNA frammenti dai nuclei in un neutro condizione1. La tecnica venne successivamente sviluppata da Singh et al., mostrando che una condizione alcalina notevolmente aumentata la specificità e la riproducibilità del test2. Da allora, l'analisi della cometa neutro principalmente è usato per rilevare le rotture del doppio filamento del DNA, mentre l'analisi alcalina della cometa è più sensibile per piccoli importi di danno del DNA, compreso il singolo e doppio filamento del DNA si rompe, siti alcali-labili, DNA-DNA o Cross-linking del DNA-proteina e rotture del singolo filamento del DNA connesse con l'asportazione incompleta ripristino siti3,4. Entrambe le analisi permettono la visualizzazione di DNA frammentato e forniscono un modo semplice per valutare quantitativamente il danno del DNA. Il test della cometa è considerato come un metodo sensibile per gli studi tossicologici genetici in vitro e in vivo ed è applicabile ad aree di ricerca diversi, quali la selezione precoce di droga-candidato, monitoraggio ambientale, biomonitoraggio umano, e fondamentale ricercare in danno del DNA e riparazione5.
Il principio del test è che, in un campo elettrico, DNA frammentato migra fuori dal corpo di nucleoide (conosciuto anche come la "testa della cometa") e forma una macchia di DNA in gel di agarosio (noto anche come "coda di cometa"). Con la colorazione del nucleotide nella misura di danno del DNA può essere quantificata analizzando "comete" formate da questo elettroforesi unicellulare. Calcolo del momento di coda può aiutare ulteriormente a confrontare il danno del DNA fra differenti gruppi sperimentali. Rispetto ai tradizionali metodi di rilevamento dei danni del DNA, il test della cometa è diretto, sensibile, poco costoso e relativamente semplice.
La radioterapia e la chemioterapia è strategie comuni per il trattamento del cancro generando filo singolo e doppio filamento DNA si rompe in cromosomi6. Il recente progresso nella inibitori di riparazione del DNA permette un più efficace effetto genotossico di chemioterapia di combinazione e quindi, potenzialmente riduce effetti collaterali sistemici quali l'anemia, infezioni e midollo osseo soppressione7, 8. in questo studio, abbiamo dimostrato l'inchiesta di un inibitore di poli (ADP-ribosio) polimerasi (PARP), olaparib (Ola)9. PARP è una proteina nucleare abbondante ed è responsabile della riparazione bassa di asportazione del DNA formando un polimero di poli (ADP-ribosio)10. Temozolomide (TMZ) è un agente alchilante orale disponibile ed è stato ampiamente usato per il trattamento di pazienti di glioma. Utilizzando l'analisi della cometa per quantificare il danno del DNA, dimostriamo che combinando olaparib con temozolomide profondamente migliora il danno del DNA in cellule del glioma, che suggerisce la terapia di combinazione di olaparib/temozolomide è una strategia efficace per il trattamento di gliomi, confrontato con temozolomide solo11.

<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

Danno del DNA è un fenomeno comune per ogni cella durante la sua esistenza ed è definito come un'alterazione della struttura chimica del DNA genomico. Terapie del cancro, come la radio e la chemioterapia, introducono una quantità enorme di ulteriori danni al DNA, che conduce all'arresto del ciclo cellulare e apoptosi per limitare la progressione del cancro. Valutazione quantitativa del danno del DNA durante la terapia sperimentale del cancro è un passo fondamentale per giustificare l'efficacia di un agente genotossico. In questo studio, ci concentriamo su un'analisi di elettroforesi unicellulare, noto anche come il test della cometa, che si può quantificare singolo e doppio filamento DNA si rompe in vitro. Il test della cometa è un metodo di quantificazione del danno del DNA che è efficiente e facile da eseguire, e ha basse esigenze di budget di tempo e un'elevata riproducibilità. Qui, si evidenzia l'utilità di analisi della cometa per uno studio preclinico valutando l'effetto genotossico olaparib/temozolomide della terapia di combinazione di cellule di glioma U251.

Il presente protocollo viene descritto un flusso di lavoro dettagliato per l'esecuzione di analisi di cometa e analisi dei dati (Figura 1). Risultati dall'analisi della cometa alcalino e neutro ha mostrato che la coda della cometa di cellule di U251 trattati con doxorubicina (1 µM, 20 h) era più lungo e aveva una maggiore intensità di DNA, suggerendo un notevole accumulo di DNA frammentato a causa della chemioterapia (Figura 2...

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Evaluating In Vitro DNA Damage Using Comet Assay

Valutazione In Vitro del DNA danni utilizzando test della cometa

Il test della cometa è un metodo efficace per rilevare danni al DNA compreso singolo e doppio filamento di DNA si rompe. Descriviamo la cometa alcalino e neutro saggi per misurare il danno del DNA in cellule di cancro per valutare l'effetto terapeutico della chemioterapia.

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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Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay

37.1K Views.  National Cancer Institute. Il test della cometa è un metodo efficace per rilevare danni al DNA compreso singolo e doppio filamento di DNA si rompe. Descriviamo la cometa alcalino e neutro saggi per misurare il danno del DNA in cellule di cancro per valutare l'effetto terapeutico della chemioterapia.

Video: 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.

Lo spostamento verso l'alto: Un semplice e flessibile In Vitro tridimensionale invasione protocollo di analisi

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

Ricerca

Ricerca sul cancro

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.

Una procedura completa per valutare le prestazioni In Vitro del Hemangioblastoma Neovascularization putativo utilizzando la sferoide germogliatura 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 ...

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

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

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.

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 ...

Biologia

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 ...

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.

Elettroforesi su gel alcalino a cellula singola: una tecnica sensibile per la stima quantitativa del danno al DNA in singole cellule tumorali dopo la chemioterapia

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

Biological Techniques

Electrophoresis Techniques

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.

Saggio della cometa per il rilevamento del danno al DNA nelle cellule

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 ...