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

Nos gustaría dar las gracias a la LRP Stiftung, en Triesen Liechtenstein, por apoyar financieramente nuestra investigación sobre el estrés oxidativo.

Estudios sobre ensayo cometa realizadas en la Universidad de Basilea, Departamento de Oftalmología fueron aprobados por el Comité Ético Local de Basilea 1. Preparación de los reactivos <ol><li> Prepare la solución mediante la adición de PBS (1 L de paquetes) y 990 ml de dH2O a los medios de comunicación Tampón fosfato salino de Dulbecco (PBS) (libre de Ca ++, Mg ++). Ajustar el pH a 7,4. Guarde a temperatura ambiente. </li><li> Preparar la solución de lisis: Para preparar 1000 ml Añadir NaCl 2,5 M (146,1 g), EDTA 100 mM (37,2 g) y 10 mM de base Trizma (1,2 g) en dH 2 O. </li><li> Preparar tampón de electroforesis (300 mM NaOH / EDTA 1 mM) mediante la adición de 30 ml de NaOH 10 M y 7,5 ml de 0,2 M EDTA, QS para 1500 ml dH 2 O y mezclar bien. El volumen total depende de la capacidad de la caja del gel. Antes de usar, medir el pH del tampón para asegurar un pH&gt; 13. </li><li> Preparar tampón de neutralización que contiene 0,4 M Tris (48,5 gm añadido a ~ 800 ml dH2O, ajustar el pH a 7,5), se concentró (&amp; #62; HCl 10 M) en 1000 ml con dH2O Guarde la solución a temperatura ambiente. </li><li> Prepare la solución de tinción. Para bromuro de etidio (EtBr; 10x Stock, 20 mg / ml) añadir 10 mg de EtBr en 50 ml dH2O y almacenar a temperatura ambiente. Para 1x Acción - mezclar 1 ml con 9 ml dH2O PRUDENTE! Maneje con precaución EtBr adecuada ya que es un carcinógeno conocido. </li></ol> 2. Aislamiento de leucocitos <ol><li> Obtener muestras de sangre humana (20 ml) con anti-coagulante tales como la heparina usando punción venosa. </li><li> Separar los linfocitos utilizando un separador de células en gradiente de densidad, tales como Histopaque. <ol><li> Brevemente, diluir la sangre en proporción 1: 1 con PBS o RPMI (sin FBS) y la capa de más de 600 l de líquido separador de células. </li><li> Centrífuga 1800 xg durante 20 min a 4 ° C. Aspirar el escudo &#39;Buffy&#39; en 3-5 ml de PBS / RPMI y se centrifugaron a 300 xg durante 10 min para sedimentar los linfocitos. </li><li> Recuperar los linfocitos desde justo por encima de límite entre PBS (RPMI) Y el líquido separador de células, utilizando una pipeta. Retire las bandas de leucocitos desde la interfaz entre el plasma y las capas líquidas separador de células de cada tubo y recoger en un tubo de 50 ml. </li></ol></li><li> Llevar el volumen total a 50 ml con medio Eagle modificado de Dulbecco frío (DMEM). Se lava la suspensión celular tres veces con DMEM a 300 xg durante 10 min. </li><li> Cuente el número total de células utilizando un hemocitómetro. Suspender las células en PBS y alícuotas en tubos de microcentrífuga a 10 7 células / tubo. </li><li> Pipetear 0,4 ml de células en un tubo de plástico desechable de 5 ml. Añadir 1,2 ml 1% de agarosa de baja temperatura de gelificación a 40 ° C. Mezclar y rápidamente y pipetear 1,2 ml de suspensión celular sobre la superficie de agarosa-cubierto de un portaobjetos pre-recubiertos. Evitar la producción de burbujas. </li><li> Deje que el gel de agarosa al por alrededor de 2 min. Sea consistente con el tiempo y la temperatura utilizada para la gelificación, y asegúrese de que agarosa está totalmente ajustado antes de sumergirse en la solución de lisis. Después plenamente set, sumergir en solución de lisis a ~ 4 ºC. </li></ol> 3. Lisis y Electroforesis NOTA: El procedimiento descrito es para la electroforesis en condiciones de pH&gt; 13 alcalinas. <ol><li> Después de al menos 2 horas a ~ 4 ºC, retire con cuidado los portaobjetos de la solución de lisis. Lugar desliza de lado a lado en la caja de gel horizontal cerca de un extremo, deslizándolas tan cerca como sea posible. </li><li> Llenar los depósitos tampón con tampón de pH&gt; 13 electroforesis recién hecho hasta que el nivel del líquido cubre completamente las diapositivas (evitar burbujas sobre la agarosa). </li><li> Deje diapositivas se sientan en el tampón alcalino durante 20 min para permitir el desenrollamiento del ADN y la expresión de daños alcalino-lábil. </li><li> Encienda la fuente de alimentación de 25 voltios (~ 0,75 V / cm) y ajustar la corriente de 300 miliamperios subiendo o bajando el nivel de amortiguación. La electroforesis los portaobjetos durante 30 min. </li><li> Desconecte la alimentación. Levante con cuidado las diapositivas de la memoria intermedia y pencaje en una bandeja de drenaje. Caída de la capa sabia las diapositivas con la neutralización de búfer. Permitir a las diapositivas para sentarse durante al menos 5 minutos. Escurrir las diapositivas y repetir dos veces más. </li><li> Teñir las diapositivas con 80 l 1x bromuro de etidio (EtBr) y dejar actuar durante 5 minutos y luego se sumerge en agua destilada fría para eliminar el exceso de tinte. A continuación, coloque un cubreobjetos sobre el portaobjetos y almacenarlos inmediatamente. Tienda desliza durante un mes a temperatura ambiente en un lugar seco. NOTA: Otras manchas de ADN (por ejemplo, SYBR-verde) puede también ser utilizado. ¡Precaución! usar guantes protectores como la mayoría de los tintes son mutagénicos. </li></ol> 4. Cuantificación de ADN Breaks <ol><li> Para visualizar el daño en el ADN, observar la diapositiva ADN teñido con EtBr bajo un microscopio de fluorescencia objetivo de 40X. </li><li> Evaluar cuantitativamente el daño del ADN en las células mediante la medición de la longitud de la migración del ADN y el porcentaje de ADN migrado (Figura 1A). Cuantificar el daño del ADN (Figura 1B) por el momento cola- y Olive-moment. Definiciones de Cola-Momento y Olive -Moment previamente se les ha dado 8-10. NOTA: En nuestra experiencia es suficiente para pegarse por uno de los parámetros. Las células son fotografiadas mediante el establecimiento de la herramienta automatizada que escanea sobre las células portaobjetos de microscopio y fotografías. Cuantificación de roturas en el ADN se puede hacer por paquetes de software de análisis de imagen. Hay varios paquetes de software diferentes que se hacen de encargo para capturar la imagen de las células y cuantificar el daño del ADN. Se recomienda utilizar el mismo paquete de software de todo el experimento. El técnico de laboratorio identifica la célula intacta o un cometa que se anotaron en el equipo y hace clic con el ratón sobre ella. La célula cometa o intacta después se anotó de forma automática. </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>Phillips, J. L., Singh, N. P., Lai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromagnetic+fields+and+DNA+damage.">Electromagnetic fields and DNA damage.</a> Pathophysiology. 16 (2-3), 79-88 (2009).</li><li>Dhawan, A., Bajpayee, M., Parmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comet+assay:+a+reliable+tool+for+the+assessment+of+DNA+damage+in+different+models.">Comet assay: a reliable tool for the assessment of DNA damage in different models.</a> Cell Biol Toxicol. 25 (1), 5-32 (2009).</li><li>Aiub, C. A., Pinto, L. F., Felzenszwalb, I. <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+genes+and+vitamin+E+in+the+prevention+of+N-nitrosodiethylamine+mutagenicity.">DNA-repair genes and vitamin E in the prevention of N-nitrosodiethylamine mutagenicity.</a> Cell Biol Toxicol. 25 (4), 393-402 (2009).</li><li>Bartsch, H., Nair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammation+and+oxidative+stress+in+the+genesis+and+perpetuation+of+cancer:+role+of+lipid+peroxidation,+DNA+damage,+and+repair.">Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair.</a> Langenbecks Arch Surg. 391 (5), 499-510 (2006).</li><li>Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+and+repair:+relevance+to+mechanisms+of+neurodegeneration.">DNA damage and repair: relevance to mechanisms of neurodegeneration.</a> J Neuropathol Exp Neurol. 67 (5), 377-387 (2008).</li><li>Choy, C. K., Benzie, I. F., Cho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-mediated+DNA+strand+breaks+in+corneal+epithelial+cells+assessed+using+the+comet+assay+procedure.">UV-mediated DNA strand breaks in corneal epithelial cells assessed using the comet assay procedure.</a> Photochem Photobiol. 81 (3), 493-497 (2005).</li><li>Mozaffarieh, M., 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=Comet+assay+analysis+of+single-stranded+DNA+breaks+in+circulating+leukocytes+of+glaucoma+patients.">Comet assay analysis of single-stranded DNA breaks in circulating leukocytes of glaucoma patients.</a> Mol Vis. 14, 1584-1588 (2008).</li><li>Mozaffarieh, M., Konieczka, K., Hauenstein, D., Schoetzau, A., Flammer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half+a+pack+of+cigarettes+a+day+more+than+doubles+DNA+breaks+in+circulating+leukocytes.">Half a pack of cigarettes a day more than doubles DNA breaks in circulating leukocytes.</a> Tob Induc Dis. 8 (14), (2010).</li><li>Mozaffarieh, M., Schotzau, A., Josifova, T., Flammer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ranibizumab+versus+photodynamic+therapy+on+DNA+damage+in+patients+with+exudative+macular+degeneration.">The effect of ranibizumab versus photodynamic therapy on DNA damage in patients with exudative macular degeneration.</a> Mol Vis. 15, 1194-1199 (2009).</li><li>Pertynska-Marczewska, M., Merhi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+Advanced+Glycation+End+Products+With+Cardiovascular+Disease+in+Menopausal+Women.">Relationship of Advanced Glycation End Products With Cardiovascular Disease in Menopausal Women.</a> Reprod Sci. , (2014).</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, 184-191 (1988).</li><li>Gaiv&#227;o, I., Sierra, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+comet+assay:+insights,+uses,+and+future+perspectives.">Drosophila comet assay: insights, uses, and future perspectives.</a> Front Genet. 5, 304 (2014).</li><li>Zhang, 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=DNA+damage+in+lens+epithelial+cells+and+peripheral+lymphocytes+from+age-related+cataract+patients.">DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients.</a> Ophthalmic Res. 51 (3), 124-128 (2014).</li><li>Huet, S. A., Vasseur, L., Camus, S., Chesn&#233;, C., Fessard, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+comet+and+micronucleus+assays+in+metabolic+competent+HepaRG+cells+to+predict+in+vivo+genotoxicity.">Performance of comet and micronucleus assays in metabolic competent HepaRG cells to predict in vivo genotoxicity.</a> Toxicol Sci. 138 (2), 300-309 (2014).</li><li>Danson, S., Ranson, M., Denneny, O., Cummings, J., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+the+comet-X+assay+as+a+pharmacodynamic+assay+for+measuring+DNA+cross-linking+produced+by+the+novel+anticancer+agent+RH1+during+a+phase+I+clinical+trial.">Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial.</a> Cancer chemotherapy and pharmacology. 60, 851-861 (2007).</li><li>Moller, P., Moller, L., Godschalk, R. W., Jones, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+reduction+of+comet+assay+variation+in+relation+to+DNA+damage:+studies+from+the+European+Comet+Assay+Validation+Group.">Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group.</a> Mutagenesis. 25, 109-111 (2010).</li><li>Chan, S. W., Chevalier, S., Aprikian, A., Chen, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+mitochondrial+DNA+damage+and+copy+number+in+circulating+blood:+a+sensitive+approach+to+systemic+oxidative+stress.">Simultaneous quantification of mitochondrial DNA damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.</a> BioMed Res Int. , 157-547 (2013).</li><li>Sanchez-Quesada, J. L., Perez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+lipoproteins+as+biomarkers+of+cardiovascular+risk+in+diabetes+mellitus.">Modified lipoproteins as biomarkers of cardiovascular risk in diabetes mellitus.</a> Endocrinol Nutr. 60, 518-528 (2013).</li><li>Gandhi, G., Kaur, G., Nisar, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+case+control+study+on+genetic+damage+in+individuals+residing+in+the+vicinity+of+a+mobile+phone+base+station.">Cross-sectional case control study on genetic damage in individuals residing in the vicinity of a mobile phone base station.</a> Electromagn Biol Med. 9, 1-11 (2014).</li><li>Cabarkapa, A., 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=Protective+effect+of+dry+olive+leaf+extract+in+adrenaline+induced+DNA+damage+evaluated+using+in+vitro+comet+assay+with+human+peripheral+leukocytes.">Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes.</a> Toxicol In Vitro. 28 (3), 451-456 (2014).</li></ol>

Científicos suecos Ostling y Johanson fueron los primeros en su campo para utilizar ensayo cometa para cuantificar el daño del ADN en las células 1. Las condiciones neutras que los dos científicos utilizaron, sin embargo, sólo se permite la detección de roturas de doble cadena de ADN. Singh et al. Más tarde adaptó el ensayo para su uso en condiciones alcalinas, que produjo una versión sensible que podría evaluar roturas de doble y de cadena simple de ADN y detectar sitios álcali-lábiles 12. Desde su desarrollo inicial, el ensayo ha sido modificado en varias etapas para que sea adecuado para la evaluación de los tipos de daño en el ADN en diferentes tipos de células 13-15. Al igual que con todas las técnicas, prestando una atención rigurosa a los detalles técnicos, es importante para obtener resultados precisos 16. Basándonos en nuestra experiencia, se obtienen mejores resultados si cada paso de la preparación metodológica solución al cometa cuantificación se realiza por expertos techn laboratorioicians. El uso de medio fresco y preparado correctamente, las técnicas adecuadas de pipeteo, la temporización exacta y (como se mencionó anteriormente en la sección de resultados) el uso de leucocitos recién preparados son esenciales para que la lisis y la electroforesis en gel para obtener resultados precisos 17. Todos los pasos individuales en este ensayo son igualmente importantes para la obtención de resultados fiables. 16 En los mejores resultados generales se obtienen si cada paso metodológico sencillo de preparación de la solución hasta la cuantificación cometa se realiza technicians.Important laboratorio experto parecen el uso de medios frescos y correctamente preparados , técnicas de pipeteo adecuadas, fecha exacta y como ya se hace referencia en la sección de resultados del uso de los leucocitos recién preparados para la lisis y la electroforesis en gel para obtener resultados adecuados del ensayo de 17. Como hacer otras metodologías, la técnica de ensayo cometa tiene su advanventajas y desventajas. Al ser un método sensible, el ensayo es vulnerable a los factores (por ejemplo, luz UV), que podría aumentar roturas en el ADN y por lo tanto afectar los resultados. Cualquier factor que puede aumentar el estrés oxidativo, excepto lo que se está investigando, se debe evitar. Los factores estresantes pueden aumentar el nivel de estrés oxidativo no sólo en los leucocitos, sino también en todos los demás medios de sangre y linfocitos. Como se mencionó anteriormente, hay muchas maneras diferentes y más directas de medir el estrés oxidativo 18,19. El ensayo cometa es uno de los muchos métodos que tiene ciertas ventajas. Estos incluyen su bajo costo relativo, el pequeño número de células requeridas (&lt;10 000 células) y así las pequeñas muestras de sangre requeridas por paciente, el período relativamente corto de tiempo requerido para cuantificar el daño del ADN en las células (aproximadamente 3 días), su sensibilidad y su extendida aplicabilidad al daño del ADN asnos en diferentes tipos de células. En el pasado hemos elegir trabajar con leucocitos ya que teníamos experiencia con estede tipo celular y era aplicable para el estudio particular previsto. Otra ventaja del ensayo cometa es que puede ser utilizado para detectar diferentes tipos y niveles de daño del ADN y por lo tanto se puede aplicar en varias otras áreas de estudios, además de estrés oxidativo, tales como estudios de reparación del ADN, ensayos de suplementación o estudios de genotoxicidad. El estrés oxidativo es ahora reconocido como jugando un papel crítico en la patogénesis de múltiples enfermedades. El método de ensayo de cometa, mientras que una de las muchas formas de medir el estrés oxidativo 18,19, es relativamente simple, versátil y barato. Cuando dominado, este método se puede utilizar en todas las áreas de la medicina en la que el estrés oxidativo juega papel 8-10,20,21.

Ensayo cometa fue desarrollado por primera vez por científicos suecos Ostling y Johanson 1. El ADN es una molécula cargada negativamente. Durante la electroforesis, los fragmentos de ADN más pequeñas, dañadas viajan más rápido hacia un ánodo cargado positivamente 2. El más pequeño de los fragmentos de ADN, más rápido su migración hacia el ánodo para formar un &quot;cometa&quot; típico con una &quot;cabeza&quot;, compuesto por intacta, el ADN dañado y una &quot;cola&quot;, compuesto de fragmentos de ADN dañadas 3. ADN dañado puede ser reparado por diferentes mecanismos en el cuerpo 4, que mantienen la generación de ADN se rompe bastante equilibradas 5. Sin embargo, si el saldo es a favor de ADN se rompe, se rompe se acumulan y en última instancia, contribuir al desarrollo de una enfermedad. Nuestro ADN sufre aproximadamente 0.000165% de daños en un momento dado 6. Breaks ADN monocatenario se refieren a un defecto en una de las dos hebras de la doble hélice de ADNmientras que las pausas de doble cadena de ADN son causadas cuando las dos cadenas de la doble hélice de ADN están dañados. Hay varios factores que pueden aumentar la cantidad de roturas en el ADN en un momento dado, como la edad de la célula, el humo del cigarrillo, diversos fármacos o el estrés oxidativo 7-9. Si se mantienen constantes todos los factores que conducen a roturas en el ADN mejoradas, a continuación, la cuantificación de ADN se rompe por medio del ensayo cometa es un buen parámetro indirecto del estrés oxidativo. El método de ensayo cometa se utiliza cada vez más hoy en la medicina incluyendo oftalmología. Una razón para esto es que el estrés oxidativo es más y más reconocido como un mecanismo patogénico para diversas enfermedades 8-11 y ensayo cometa es un método con el que el estrés oxidativo se puede cuantificar indirectamente.

<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

Organismos eucariotas superiores no pueden vivir sin oxígeno; sin embargo, paradójicamente, el oxígeno puede ser perjudicial para ellos. La molécula de oxígeno es químicamente relativamente inerte porque tiene dos electrones desapareados ubicados en diferentes orbitales pi * anti-unión. Estos dos electrones tienen espines paralelos, lo que significa que giran en la misma dirección alrededor de sus propios ejes. Esta es la razón por la molécula de oxígeno no es muy reactivo. La activación de oxígeno puede ocurrir por dos mecanismos diferentes; ya sea a través de la reducción a través de un electrón en un momento (reducción monovalente), o mediante la absorción de energía suficiente para invertir el giro de uno de los electrones desapareados. Esto resulta en la producción de especies oxidativas reactivas (ROS). Hay un número de maneras en que el cuerpo humano elimina ROS en su estado fisiológico. Si la producción de ROS supera la capacidad de reparación, los resultados de estrés oxidativo y los daños de diferentes moléculas. Hay muchos métodos diferentes por los que el estrés oxidativo puede ser míasured. Este manuscrito se centra en uno de los métodos llamados electroforesis en gel de célula, también conocido como &quot;ensayo cometa&quot;, que permite la medición de roturas en el ADN. Si se mantienen constantes todos los factores conocidos para causar daño en el ADN, que no sea el estrés oxidativo, la cantidad de daño en el ADN medido por el ensayo de cometa es un buen parámetro de estrés oxidativo. El principio es simple y se basa en el hecho de que las moléculas de ADN están cargadas negativamente. Una molécula de ADN intacto tiene un tamaño tan grande que no migra durante la electroforesis. Roturas en el ADN, sin embargo, si está presente resultado en fragmentos más pequeños que se mueven en el campo eléctrico hacia el ánodo. Los fragmentos más pequeños migran más rápido. Como los fragmentos tienen diferentes tamaños el resultado final de la electroforesis no es una línea distinta, sino más bien un continuo con la forma de una cometa. El sistema permite una cuantificación de la &quot;cometa&quot; resultante y por lo tanto de las roturas en el ADN en la célula.

Aunque el método de ensayo cometa hace resultados reproducibles, puede ser influenciada por una variedad de factores. Uno de estos factores es si o no los leucocitos son crioconservados antes de la lisis y la electroforesis en gel o si este paso se lleva a cabo en los leucocitos recién preparados. La Figura 2 muestra que en el Grupo 2, donde los leucocitos fueron crioconservados antes de la lisis de ADN y electroforesis en gel de SS- roturas fueron significativamente más altos, que en el Grupo 1, donde la lisis y gel -electrophoresis se realizó en las células recién preparadas. El ensayo también se puede utilizar para evaluar indirectamente el estrés oxidativo sistémico. La Tabla 1 muestra los estadísticos descriptivos de cola- momento y momento oliva en fumadores y sujetos sanos. Los datos revelan que fumar medio paquete de cigarrillos al día más que duplica descansos ssDNA en los leucocitos circulantes 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) Representa el microscopio utilizado durante el análisis de ensayo cometa. (B) Δ representa a un &quot;cometa&quot; típico con una cabeza brillante y la cola (dañado fragmentos de ADN más pequeños). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52763/52763fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52763/52763fig2.jpg" /> Figura 2. rompe ssDNA fueron significativamente más altos cuando los leucocitos fueron crioconservados (Grupo 2) que cuando se utilizaron los leucocitos recién preparadas antes de la lisis y la electroforesis en gel. <imgalt = &quot;Tabla 1&quot; src = &quot;/ files / ftp_upload / 52763 / 52763table1.jpg&quot; /&gt; Tabla 1. Los fumadores tenían más del doble del número de ssDNA rompe en comparación con la edad y el sexo emparejado sanos no fumadores.

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

Ensayo cometa mide roturas en el ADN, inducidas por diferentes factores. Si se mantienen constantes todos los factores (excepto el estrés oxidativo) que causan daño en el ADN, la cantidad de daño en el ADN es un buen parámetro indirecto de estrés oxidativo. El objetivo de este protocolo es usar ensayo cometa para la medición indirecta de estrés oxidativo.

Ensayo Cometa como una medida indirecta de estrés oxidativo sistémico

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

Universidad San Sebastian

Research

JoVE Journal

Biology

29.3K vistas.  University of Basel.  Ensayo cometa mide roturas en el ADN, inducidas por diferentes factores. Si se mantienen constantes todos los factores (excepto el estrés oxidativo) que causan daño en el ADN, la cantidad de daño en el ADN es un buen parámetro indirecto de estrés oxidativo. El objetivo de este protocolo es usar ensayo cometa para la medición indirecta de estrés oxidativo. 

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.

Evaluar In Vitro el daño de ADN utilizando el ensayo 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 ...

Investigación

Investigación sobre el cáncer

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: Un gran procesamiento Plataforma 96-Bueno, para medir el daño del ADN en las células en micromatriz Humanos

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

Bioingeniería

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 ensayo de conducta para medir la capacidad de respuesta de pez cebra a cambios en la intensidad de luz

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 ...

Biología

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 de tejido congelado en el ensayo de cometas para la evaluación del daño causado por el ADN

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 ...

Genética

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.

Ensayo de daño del ADN en las neuronas del hipocampo utilizando el ensayo cometa

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 ...

Neurociencias

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

La aplicación de un<em&gt; In vitro</em&gt; DNA ensayo de protección de visualizar Estrés Mediación propiedades de la proteína Dps

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

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.

En Vivo alcalina Comet Assay y Comet Assay alcalina modificada con enzimas para la medición de roturas en el ADN Strand y daño oxidativo del ADN en el hígado de rata

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 enfoque de ensayo cometario de alto rendimiento para evaluar el daño del ADN celular

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

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.

Ensayo Comet para la detección de daños en el ADN en células

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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.

Ensayo Cometa como una medida indirecta de estrés oxidativo sistémico

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