Este trabajo fue financiado por los Institutos Nacionales de Salud (CA142664).

1. La detección de ROS con carboxi-H 2 DCFDA Carboxi-H 2 DCFDA no es fluorescente, pero en presencia de ROS, cuando este reactivo se oxida, se convierte en verde fluorescente. <ol><li> Inmediatamente antes de su uso, preparar una solución de frescos de carboxi-H 2 DCFDA en dimetilsulfóxido estéril (DMSO) o el 100% de etanol. Evitar la repetición descongelar / congelar los ciclos de su tinte. </li><li> Se lavan las células con solución salina tamponada con HEPES (HBSS) o tampón fosfato salino (PBS) para eliminar los restos del medio original. </li><li> La carga de las células con el colorante (y el tinte de control, véase el punto la nota). En este ensayo hemos utilizado Jurkat, una línea celular de leucemia humana. Use carboxi-H 2 DCFDA a una concentración final de 1μM en medio de cultivo regular con suero reducido (2%). </li><li> Incubar los cultivos durante 30 minutos en la oscuridad, en una incubadora convencional (37 ° C, 5% CO2). Descartar las soluciones colorantes utilizados. </li><li> Quitar carboxi-H 2 DCFDA que contienen medio y lavar dos veces con HBSS o PBS. A partir de este paso adelante, a proteger las células de la luz. </li><li> Añadir un nuevo soporte de la droga de elección para la carboxi-H 2 DCFDA células cargadas e incubar como se desee. Para este ejemplo vamos a usar H 2 O 2 (0,03%) durante 1 hora. </li><li> ROS evaluar de inmediato el análisis de las células por citometría de flujo a través del canal FL1 (fluorescencia verde), o mediante un lector de placas de fluorescencia, o por microscopía de fluorescencia. La fluorescencia se puede detectar mediante el uso de longitudes de onda de excitación y emisión que son apropiados para la fluorescencia verde. </li></ol> Nota: Los controles deberán incluir carboxi-H 2 DCFDA cargado las células no tratadas y teñidas las células no tratadas. Carboxi-H 2 DCFDA se conoce para detectar peróxidos, pero también podría ser oxidado por otros ROS. Este reactivo también puede ser modificado por otros medios, colorantes de oxidación de control insensible como el 5 - (y-6)-carboxi-2 &#39;, 7&#39;-diclorodiacetato ofluorescein (carboxi-DCFDA) por lo tanto, deben incluirse en la prueba. 2. La medición del óxido nítrico (NO) Usted tendrá que sulfanilamida y N-1-napthylethylenediamine soluciones dihidrocloruro (NED), y el nivel de nitritos. Este ensayo se denomina ensayo de Griess. Solución NED: Haga una solución al 0,1% de diclorhidrato de N-1-napthylethylenediamine diluida en solución estéril water.Sulfanilamide: Haga una solución al 1% de sulfanilamida diluido en ácido fosfórico al 5%. Estándar de nitrito: Diluir el 0,1 M de patrón de nitrito de sodio a 100μM en medio estéril, hacer una serie de dilución en el mismo medio. Condiciones de almacenamiento: Almacene los productos químicos según las instrucciones del fabricante a temperatura ambiente. Una vez reconstituido, la NED y las soluciones de sulfanilamida se almacenan inmediatamente después de su uso a 4 ° C, en la oscuridad, y por un máximo de 3 meses. <ol><li> Las células de cultivo en una placa de 96 pocillos,uso por triplicado para cada condición, e incluyen controles adecuados de acuerdo con sus experimentos. </li><li> Tratamiento de las células para inducir la producción de NO. En nuestro experimento utilizamos lipopolisacárido (LPS) (100 ng / ml) e IL-4 recombinante para el tratamiento de las células. En este protocolo, el RAW 264.7, una línea celular de macrófagos de ratón (mouse leucémicas línea celular de macrófagos monocitos). </li><li> En el día de la determinación, tanto de los reactivos a temperatura ambiente. </li><li> Girar el plato, recoger los sobrenadantes celulares y la transferencia de 50μl con una nueva placa de 96 pocillos. Prepare limitar los pozos de dilución de la solución madre de patrón para hacer una curva estándar. </li><li> Añadir 50μl de la solución de sulfanilamida a cada muestra y control, así y mezclar bien. </li><li> Incube a temperatura ambiente durante 10 minutos en la oscuridad. </li><li> Añadir 50μl de solución de clorhidrato de N-1-napthylethylenediamine a cada muestra y control, así y mezclar bien. </li><li> Incube a temperatura ambiente durante 10 minutos en la oscuridad. </li><li> Medida Absorbana inmediatamente con un lector de placas con filtro de 520 nm de longitud de onda entre 550 nm y. </li></ol> Si usted utiliza diferentes plato / plato de tamaño, el uso 1/1/1 volumen para cada solución y el sobrenadante de la muestra. Un color violeta aparecerá en los pozos positivos. Los resultados obtenidos con el estándar le ayudará a comprobar la estabilidad de sus soluciones. 3. La detección de ROS acción: proteínas oxidadas Un método diferente para identificar la producción de ROS es ver el resultado final mediante la detección de la oxidación de las proteínas. De hecho, ROS modificar el glutatión, un antioxidante que se expresa en la mayoría de las células y juega un papel protector contra las ROS. Después de la oxidación por ROS, la modificación del glutatión reducido (GSH) se traduce en la sulfhidrilo (tiol) del grupo de la cisteína estar vinculado a un glutatión segundo a través de un puente disulfuro. Esto lleva a la formación de una proteína dimerizados (GSSG oxidada proteína). GSH puede ser restaurada a través de la modificación de GSSG por la enzima glutatión reductasa. El aumento en el cociente GSSG / GSH refleja el estrés oxidativo. El siguiente ensayo se basa en la detección y cuantificación de estas proteínas oxidadas. Este método no es selectivo para ROS específicos, sino más bien detecta los efectos del NO, H 2 O 2, O 2 - y otros ROS. Aquí se mide la cantidad total de oxidado (GSSG) y reducido (GSH) glutatión con señales bioluminiscentes. <ol><li> Semillas de las células en una placa de 96 pocillos (blanco / transparente, fondo plano), como de costumbre. Para nuestro experimento, se utilizó 1x10 4 células por pocillo de adherentes y Raw 264.7 A549 y células Jurkat suspensión. Dependiendo del tamaño de sus células estos números podrían ser ajustados. Recomendamos a las células de la placa adherida al día antes de la prueba que les permita conectar a la placa. Suficientes pozos deben estar preparados para la detección de proteínas oxidadas y reducidas, así como de &quot;único medio&quot; ylas células no tratadas como controles. Use triplicado en todas las condiciones. </li><li> Tratar las células con su medicamento durante el tiempo necesario. Aquí se tratan las células Jurkat y las células A549 durante 1 hora con H 2 O 2 (a 5 mm y 2,5 mm respectivamente). Raw 264,7 células fueron tratadas con 200ng/ml de LPS durante 16 h. Durante este tiempo de incubación, llevar a todos los reactivos a temperatura ambiente (TA). Hacer todos los reactivos no más de 30 minutos antes de realizar la prueba. La tabla siguiente muestra los volúmenes de los reactivos por pozo. Cuando sea posible, es importante para eliminar los medios de comunicación que contiene el agente reductor antes de proceder con la prueba. </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> Células adherentes </td><td colspan="2"> Células en suspensión </td></tr><tr><td> Lisis total de Glu </td><td> Oxidado Glu Lisis </td><td> Lisis total de Glu </td><td> Oxidado Glu Lisis </strong&gt; </td></tr><tr><td> NEM, 25 mM </td><td> ninguno </td><td> 0.5μl </td><td> ninguno </td><td> 0.5μl </td></tr><tr><td> Luciferina-NT </td><td> 1 l </td><td> 1 l </td><td> 1 l </td><td> 1 l </td></tr><tr><td> Tampón de lisis pasiva, 5X </td><td> 10μl </td><td> 10μl </td><td> 10μl </td><td> 10μl </td></tr><tr><td> Agua destilada </td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> El volumen final por pocillo </td><td> 50μl </td><td> 50μl </td><td> 25μl </td><td> 25μl </td></tr></tbody></table><ol start="3"><li> Quitar medio de pozos con células adherentes. No quite medio de pozos con células en suspensión. </li><li> Añadir el reactivo reducido lisis glutatión glutatión oxidado o reactivo de lisis de los pozos correspondientes y agitar durante 5 minutos a temperatura ambiente. </li><li> Añadir el reactivo luciferina y la generación de incubate durante 30 minutos a temperatura ambiente. </li><li> Añadir el reactivo de detección luciferina y se incuba durante 15 minutos a temperatura ambiente. </li><li> Lea la señal bioluminiscente en el momento de la integración de 0,25-1 por segundo y con un luminómetro lector de placas. </li></ol><table border="1"><tbody><tr><td></td><td> Células adherentes </td><td> Células en suspensión </td></tr><tr><td> El número de células por pozo </td><td> 1x10 4 </td><td> 1x10 4 </td></tr><tr><td> De células en suspensión por drogas y + </td><td> 100 l, para ser removido antes de 3,4 </td><td> 25μl, para no ser eliminado </td></tr><tr><td> El glutatión reducido reactivo de lisis </td><td> 50 l </td><td> 25μl </td></tr><tr><td> Reactivo de lisis glutatión oxidado </td><td> 50 l </td><td> 25μl </td></tr><tr><td> Luciferina reactivo generación </td><td> 50 l </td><td> 50 l </td></tr><tr><td> Luciferina reactivo de detección </td><td> 100 l </td><td> 100 l </td></tr></tbody></table> 4. Los resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> Figura 1 La detección de ROS con carboxi-H2DCFDA tinte. Células Jurkat (línea celular humana de leucemia) tratados con H 2 O 2 se compararon con las células no tratadas. ROS induce la modificación de carboxi-H 2 DCFDA que verde fluorescente detectada por citometría de flujo, el pico fluorescente H 2 O 2 células tratadas cambio en comparación con los picos en los controles (H 2 O 2 células tratadas teñidas con colorantes de oxidación insensible y no tratados con las células teñidas con carboxi-H 2 DCFDA). Los resultados confirman la presencia de ROS en las células tratadas. <img alt="Figura 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> Figura 2 Detección of NO utilizando reactivos de Griess. RAW 264.7 (macrófago de ratón) fueron tratados con LPS e IL-4. Un aumento significativo en la producción de NO se detectó en las células tratadas en comparación con el control de las células no tratadas. <table border="1"><tbody><tr><td> Las líneas celulares </td><td> No tratados </td><td> Tratados </td></tr><tr><td> Raw 264,7 </td><td> 13.0 </td><td> 8.3 </td></tr><tr><td> A549 </td><td> 21.6 </td><td> 10.5 </td></tr><tr><td> Jurkat </td><td> 5.2 </td><td> 2.8 </td></tr></tbody></table> Tabla 1 La detección de ROS mediada por la oxidación de las proteínas. RAW 264,7 células fueron tratadas con LPS, Jurkat y A549 (cáncer de pulmón humano), las células fueron tratadas con H 2 O 2. Los resultados se expresan como el cociente reducido (GSH) / oxidado (GSSG) glutatión. Cocientes más bajos de glutation (GSH) / (GSSG) se detectaron en las células tratadas en comparación concontrol de las células no tratadas, dejando al descubierto que las proteínas eran más oxidados en las células tratadas.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

Hemos recibido el apoyo de Promega para esta publicación.

Varias situaciones patológicas, como enfermedades inflamatorias, cáncer, isquemia / reperfusión, y también tratamientos como la radioterapia o la quimioterapia (cisplatino por ejemplo) induce una sobreproducción de ROS. Por lo tanto, la detección y medición de los niveles de ROS es importante en muchos estudios básicos, pre-clínicos y clínicos. Sin embargo, ROS tienen una vida media muy corta y puede ser complicado de detectar. Aquí, proponemos pruebas simples que se utilizan habitualmente y ampliamente acept...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> 5 - (y-6)-carboxi-2 &#39;, 7&#39;-diacetato diclorofluoresceína (carboxi-DCFDA) </td><td> Las sondas moleculares </td><td> C369 </td><td> control </td></tr><tr><td> carboxi-H 2 DCFDA </td><td> Las sondas moleculares </td><td> C400 </td><td></td></tr><tr><td> Sulfanilamida </td><td> Sigma </td><td> S9251-100G </td><td></td></tr><tr><td> N-1-napthylethylenediamine dihidrocloruro </td><td> Sigma </td><td> N9125-10G </td><td></td></tr><tr><td> Nitrito estándar </td><td> Sigma </td><td> 237213-100G </td><td></td></tr><tr><td> GSH / GSSG-Glo ensayo </td><td> Promega </td><td> V6612 </td><td> Para cuantificar oxidado, reducido u oxidado / glutatión reducido </td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

Las especies reactivas del oxígeno incluyen una serie de moléculas que dañan el ADN y ARN, y se oxidan las proteínas y los lípidos (peroxidación lipídica). Estas moléculas reactivas contienen un átomo de oxígeno y son H 2 O 2 (peróxido de hidrógeno), NO (óxido nítrico), O 2 - (anión), peroxinitrito (ONOO -), ácido hydrochlorous (HOCl), y el radical hidroxilo (OH -) . Especies reactivas de oxígeno se producen no sólo en situaciones patológicas (cáncer, isquemia reperfusión /, patologías neurológicas y cardiovasculares, enfermedades infecciosas, enfermedades inflamatorias 1, 2 enfermedades autoinmunes, etc ...), sino también en situaciones fisiológicas (no patológicas), tales como el metabolismo celular 3 , 4. De hecho, ROS juegan un papel importante en muchas vías de señalización celular (proliferación, activación de las células 5, 6, 7 migración, etc.). ROS puede ser perjudicial (entonces se conoce como&quot;El estrés oxidativo y nitrosativo&quot;) cuando se produce en grandes cantidades en los compartimentos intracelulares y las células responden en general a ROS por upregulating antioxidantes como la superóxido dismutasa (SOD) y catalasa (CAT), glutatión peroxidasa (GPx) y glutatión (GSH), que protege ellas mediante la conversión de los peligrosos radicales libres a las moléculas inofensivas (por ejemplo, agua). Las vitaminas C y E también se han descrito como basureros ROS (antioxidantes). Los radicales libres son beneficiosos en pequeñas cantidades 3. Macrófagos y neutrófilos respuestas inmunes mediadas por involucrar a la producción y liberación de NO, que inhibe virus, patógenos y la proliferación del tumor 8. El NO también reacciona con otros ROS y por lo tanto, también tiene un papel como un desintoxicante (scavenger ROS). Por último, no actúa sobre los vasos para regular el flujo de sangre que es importante para la adaptación del músculo al ejercicio prolongado 9, 10. Varias publicaciones han demostrado también que las ROS participan en sens insulinapositividad 11, 12. Numerosos métodos para evaluar la producción de ROS se encuentran disponibles. En este artículo nos proponemos varias pruebas sencillas, rápidas y asequibles, estos ensayos han sido validadas por numerosas publicaciones y se usan rutinariamente para detectar ROS o sus efectos en células de mamíferos. Aunque algunos de estos ensayos de detección de múltiples ROS, otros detectan un solo ROS.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

Aquí proponemos métodos sencillos para probar y evaluar la presencia de especies reactivas del oxígeno en las células.

Producción y la detección de especies reactivas del oxígeno (ROS) en los cánceres

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.8K vistas.  Baylor College of Medicine. Aquí proponemos métodos sencillos para probar y evaluar la presencia de especies reactivas del oxígeno en las células.

Video: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualización y análisis de flujo de la sangre y del consumo de oxígeno en la microcirculación hepática: Aplicación a un Modelo de cuadro de hepatitis aguda

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Investigación

Medicina

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Cerenkov Luminiscencia Imaging (CLI) para la Vigilancia del Cáncer Terapia

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

El aislamiento y uso de las Secciones de bovino mesentérica arteria y la vena como un bioensayo para la Prueba de Vasoactividad en el intestino delgado

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

La evaluación de la encefalopatía espongiforme transmisible Especies Barreras con un<em&gt; In Vitro</em&gt; Prion Análisis de Conversión de proteínas

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

La detección de la enfermedad asociada-α-sinucleína por ELISA mejorada en el cerebro de ratones transgénicos que sobreexpresan A53T humano mutado α-sinucleína

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Disparo reactiva Gliosis<em&gt; En Vivo</em&gt; Por un Prosencéfalo Lesiones Puñalada

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo la detección de la mitofagia en células humanas, C. Elegansy los ratones

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Detección basada en plaquetas de óxido nítrico en sangre mediante la medición de VASP fosforilación

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Mapeo cuantitativo de ventilación específica en el pulmón humano usando resonancia magnética de protones y oxígeno como agente de contraste

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

Caracterización de la enfermedad durante la desoxigenación automatizada controlada con ektacytometría de gradiente de oxígeno

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...