This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. Calibración óptica: Medición de luz de salida <ol><li> Para preparar el aparato de suministro de luz, conectar una fuente de luz de banda ancha (por ejemplo, xenón o lámpara de tungsteno) a una fuente de alimentación adecuada. Colocar una lente de colimación en frente de la fuente de luz para producir un haz de luz colimado. Pasar la luz a través de un filtro de calor líquido para eliminar la mayor parte del calor del haz de luz. Dependiendo de la aplicación, enfocar el haz colimado a la abertura de entrada de una guía de luz líquida, que proporciona para la entrega más flexible de la luz (por ejemplo., En una incubadora). </li><li> En el otro extremo de la guía de luz líquida, posicionar una segunda lente de colimación y, a continuación un soporte para la interferencia y filtros de densidad neutra. Compruebe que esta disposición produce una mancha uniformemente iluminada de luz de la banda de frecuencia y la intensidad deseada. Nota: La distancia entre el extremo de la guía de luz y el plano de la muestra puede tener que variar dependiendoel área que debe ser iluminada, pero recuerda que como la distancia desde el extremo de la guía de luz aumenta, intensidad de la luz disminuye. En el presente estudio, esta distancia es de 14cm y produce una sección transversal del haz que ilumina uniformemente un área bien 3x3 en una placa de 96 pocillos. </li><li> Asegúrese de que la luz difusa de la lámpara y los componentes ópticos asociados no puede llegar a la muestra. </li><li> Encienda la fuente de agua para el filtro de calor líquido y asegurar que existe intercambio de agua a través del filtro de la chaqueta. Encienda la fuente de energía de la lámpara y espere por lo menos 5 minutos para la fuente de luz de banda ancha a estabilizarse. Nota: Se requiere el uso del enfriador de agua para evitar el sobrecalentamiento del dispositivo. </li><li> Seleccionar un filtro de interferencia de banda estrecha (por lo general descrito por su longitud de onda central, la transmitancia de pico y anchura total a la mitad del máximo (FWHM) de ancho de banda) para generar la banda de frecuencias deseada de la iluminación. Nota: Los filtros de interferencia de banda estrecha utilizadas en el estudio fueron 442 nm, 550 nm, 671 nm y 810 nm. </li><li> Medir la luz producida por la lámpara en el plano en que el espécimen se va a colocar durante el tratamiento. Medir la luz utilizando una sonda calibrada irradiancia (colector coseno) conectado a un espectrorradiómetro adecuado, el uso de software de propiedad de acuerdo con las instrucciones del fabricante. </li><li> Utilice filtros de densidad neutra para ajustar la intensidad de la luz hasta que se obtiene la salida deseada. Nota: En el presente estudio, la intensidad de luz que pasa por cada filtro de interferencia se ajusta para dar una salida quantal igual en cada una de las cuatro bandas de frecuencia de tratamiento. Es importante comprobar la calibración con regularidad, como la salida de la lámpara está sujeta a cambios. Nota: Tras la puesta en marcha del aparato, las células están listas para ser iluminada Figura 1 es una representación del aparato de suministro de luz utilizada en el estudio actual.. </li></ol><img alt="Figura 1"src = &quot;/ files / ftp_upload / 52221 / 52221fig1.jpg&quot; /&gt; Figura 1. Imagen del aparato de suministro de luz. Illustrated son la fuente de energía de luz, lámpara de xenón con la vivienda, la lente de colimación, filtro de agua, la abertura de entrada, guía de luz líquida, segunda lente de colimación, soporte de filtro, marco de tratamiento y mate tarjeta de negro. Tenga en cuenta que los filtros de longitud de onda e intensidad de banda estrecha no se muestran. 2. Preparación de la célula <ol><li> El método de entrega / NIR-LT R descrito puede ser aplicado a cualquier tipo de célula o en el sistema de modelo in vitro; como tal, las siguientes descripciones de cultivo celular son generales, usando técnicas bien establecidas para la cultura feocromocitoma (PC12), Müller (RMC1) y células de la retina mixtos primarios. </li><li> Antes de la siembra de células para el tratamiento de la luz y el ensayo de estrés oxidativo, la cultura células en sus respectivos medios de cultivo que contiene suplementos apropiados (por ejemplo., FBS, antibióticos) en flas T75 inmortalizóks hasta 70-80% de confluencia. </li><li> Separar las células inmortalizadas de los matraces T75, usando el método apropiado para el tipo de célula para ser probado por ejemplo., Tripsina. Si es necesario, antes de proceder con la siembra de células en placas de 96 pocillos de ensayo, pocillos de placas de ensayo de capa con 10μg / ml de poli-L-lisina durante 1 h (por ejemplo., Para las células PC12) o 10μg / ml de poli-L-lisina para 1H seguido de un / O N incubación con 10μg / ml de laminina (por ejemplo., por células de la retina mixtos). </li><li> Centrifugar las células a recoger en su caso (por ejemplo., A 3 minutos a 405 xg para las células PC12 o 10 min a 218 xg durante células RMC1), eliminar el sobrenadante y resuspender en 8 ml de medio de cultivo celular apropiado. </li><li> Si las células de la retina son mezclados para ser utilizado, preparar a partir de crías de rata neonatal P0-5 por digestión enzimática (papaína) de acuerdo con procedimientos establecidos 19 </li><li> Contar el número de células viables con exclusión de 0,4% (w / v) de colorante azul de tripano, usando un hemocitómetro. </li><li> Ajuste suc densidad celularh que las células serán de aproximadamente 70-80% de confluencia después de 24 o 48 horas en cultivo. (Para las células PC12 de la densidad de siembra es de 4,0 x 10 5 células viables / ml, para las células RMC1, es 2,5 x 10 5 células viables / ml y para células de la retina mixtos es 8 x 10 5 células viables / ml). Después de añadir las células ya sea a placas de 96 pocillos claros o negros (utilizados para el H 2 O 2 o ensayo DCFH-DA, respectivamente), en 100 l de medio de cultivo adecuado, permiten placa para sentarse en una superficie plana a 37 ° C, 5 % de CO 2 (o lo que las condiciones son apropiadas para el tipo específico de célula) durante al menos 24 horas para permitir la adherencia celular. </li><li> Cultivar las células en medio de crecimiento en las 96 bandejas así apropiadas hasta que son 70-80% de confluencia (24h para PC12 y las células RMC1, 48h para las células de la retina mixtos). </li></ol> 3. La adición de glutamato Stressor a células <ol><li> Preparar monosódico ácido concentraciones estresantes hidrato de sal de L-glutámico de 0-10mm en cultivo completo apropiadomedios de cultivo XX. </li><li> Retire el medio de las células y lavar suavemente las células 3 veces con PBS (PC12) o HBSS (RMC1 y células de la retina mixtos). Después de lavados, añadir medios que contienen glutamato a un volumen total de 100 l / pocillo. </li></ol> 4. Primera Dosis de Tratamiento Luz <ol><li> Inmediatamente después de la adición de glutamato o el factor estresante de elección, exponer las células a tratamiento con luz en las longitudes de onda e intensidades deseadas colocando bajo el aparato de suministro de luz preparada. Asegúrese de alterar la salida quantal del haz de luz usando las combinaciones establecidas de filtros de densidad neutra en función de la longitud de onda que se administra. Nota: En los experimentos actuales, exponer las células para el tratamiento de luz durante 3 min mantener la temperatura en reposo las placas de 96 pocillos en una almohadilla de calor C 37 °. El tiempo de tratamiento puede variar según se requiera. <ol><li> Coloque una tarjeta mate negro debajo de las células para evitar que la luz refleja en los pozos adyacentes en el 96-well bandeja designado para otros parámetros de tratamiento. </li></ol></li><li> Si se desea un tratamiento para longitudes más largos de tiempo, añadir tampón Hepes 25 mM para mantener el pH del medio de cultivo celular o, idealmente, colocar las placas de aparatos y de tratamiento dentro de una incubadora con CO 2 concentración regulada a 5% de CO 2, o condiciones que son óptimas para el tipo específico de célula. </li><li> Tras la finalización del tratamiento con luz, colocar las células en una superficie plana y se incuba a 37 ° C y 5% de CO 2 (o lo que las condiciones son óptimas para el tipo de célula) durante 24 horas. Nota: las dosis adicionales de R / NIR-LT se pueden administrar como se ha descrito anteriormente, según se desee. </li></ol> 5. Final de la dosis de tratamiento con luz y detección de ROS <ol><li> Antes de realizar la ronda final del tratamiento de la luz, se preparan los reactivos que se utilizan para la detección de ROS. Prepare el buffer de 50 mM de citrato (pH 6,0), Triton X100, y H 2 O 2 reactivo de detección de trabajosolución (1: 2: 97 proporción de 10 mM H 2 O 2 de detección de solución de reactivo de valores; 10 U / ml de peroxidasa de rábano; citrato de sodio 50 mM (pH 6,0)). Preparar DCFH-DA a una concentración final de 100μM en el medio adecuado. Nota: DCFH-DA reactivo para las células PC12 se encuentra en medio RPMI, DCFH-DA reactivo para células RMC1 está en medio DMEM. Tenga en cuenta que los reactivos de detección disponibles en el mercado tienen sensibilidades diferenciales a lo específico ROS y los reactivos deben ser elegidos cuidadosamente para proporcionar la información deseada para una aplicación individual. </li><li> Administrar tratamiento con luz a las células, como se describe en el paso 4.1-4.1.2. Asegurarse de que las células están siendo tratados con los deseados influencias / longitudes de onda de la luz. </li><li> Inmediatamente después de tratamiento con luz, retire los medios que contienen glutamato y lavar dos veces con solución tampón apropiado (para las células PC12, PBS y se utiliza para las células RMC1, se utiliza HBSS). Realizar los ensayos de ROS en las células como sigue: <ol><li> H 2 O 2 </sub&gt; ensayo: añadir 45 l de tampón de citrato 50 mM (pH 6,0) y 5 l Triton X100 a cada uno de los pocillos. Agite suavemente las células en un agitador orbital durante 30 segundos y se incuba a 37 ° C y 5% de CO2 durante 15 min. Añadir 50 l de H 2 O 2 solución de trabajo de detección y se incuba durante 30 min a TA. Medir la fluorescencia usando un lector de placa con una longitud de onda de excitación de 530 nm y una longitud de onda de emisión de 480 nm. </li><li> Ensayo de DCF: añadir 100 l de la solución de DCFH-DA a cada uno de los pocillos 100 mM y se incuba durante 30 minutos a 37 ° C y 5% de CO 2. Retire el medio que contiene 100 mM DCFH-DA y lavar con solución de tampón apropiado (como se describió anteriormente) dos veces. Añadir 90 l de solución tampón y 10 l Triton X100 a cada uno de los pocillos y agitar suavemente en un agitador orbital durante 30 segundos antes de 15 min de incubación a 37 ° C. Mida DCF fluorescencia derivada usando un lector de placas con una longitud de onda de excitación de 480 nm y una emisión wavelength de 530 nm. </li><li> Expreso ROS valora con respecto a la concentración de proteína de las células restantes en los pocillos, utilizando un kit colorimétrico para cuantificar la concentración de proteína de acuerdo con las instrucciones del fabricante, con referencia a una curva estándar para calcular mg de proteína. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Hemos adaptado con éxito un sistema de suministro de luz precisa y calibrado para proporcionar un mecanismo para el estudio de optimización de R / NIR-LT in vitro. Longitud de onda y la intensidad de los parámetros de R / NIR-LT son capaces de ser manipulado con precisión y eficacia el uso de este sistema. Hemos establecido que el tratamiento de luz de las células no dio lugar a la muerte celular, aunque ROS no se reduce a las longitudes de onda y las dosis entregadas, de los tipos celulares de la prueba. ...

Se requieren especies reactivas de oxígeno (ROS) para una serie de vías de transducción de señales y reacciones normales del metabolismo celular, incluidos los de neuroprotección 1. Sin embargo, cuando el mecanismo antioxidante endógeno no son capaces de controlar la producción de ROS, las células pueden sucumbir a estrés oxidativo 2,3. Después de una lesión en el SNC, se cree que los aumentos asociados en la presencia de ROS y el estrés oxidativo a desempeñar un papel importante en la progresión del daño 4,5. A pesar de la extensa serie de estrategias para atenuar el estrés oxidativo que se han evaluado, actualmente no existen estrategias de anti-oxidantes completamente eficaces, clínicamente relevantes para la atenuación de la producción de ROS y el estrés oxidativo asociado en uso clínico siguiente neurotrauma 6. Por lo tanto la atenuación del estrés oxidativo sigue siendo un objetivo importante para la intervención terapéutica 7. Mejoras siguening R / NIR-LT se han reportado en una amplia gama de lesiones y enfermedades, incluyendo reducciones en el tamaño del infarto de miocardio, las complicaciones renales y hepáticas durante diabetes, degeneración de la retina, la lesión del SNC y accidente cerebrovascular 8, quizás al reducir el estrés oxidativo. Con especial atención a la lesión del SNC, los estudios preclínicos de eficacia de la luz 670 nm han demostrado buenos efectos en los modelos de degeneración retiniana 9-11, lesión de la médula espinal 12, la muerte neuronal 13. Se han llevado a cabo ensayos clínicos para la edad seco degeneración macular relacionada y están actualmente en curso para el accidente cerebrovascular 14, sin embargo los resultados de estos ensayos no parecen prometedores, quizás debido a la imposibilidad de emplear un tratamiento efectivo parámetros 15. Como tal, R / NIR-LT no ha sido ampliamente adoptado como parte de la práctica clínica normal en neurotrauma, a pesar de ser una fácil de administrar, no invasiva y tratamiento relativamente barato. Las barreras a la traducción clínica incluyen la falta de un cltemprano entendido mecanismo de acción y la ausencia de un protocolo de tratamiento efectivo 16,17 estandarizado. La literatura actual con respecto a la terapia de luz revela una gran cantidad de variación en los parámetros de tratamiento con respecto a las fuentes de irradiación (LED o láser), longitud de onda (por ejemplo, 630, 670, 780, 810, 830, 880, 904nm), la dosis total (julios de irradiación / unidad de superficie), la duración (tiempo de exposición), el tiempo (insulto previo o posterior), la frecuencia del tratamiento y el tipo de parto (pulso o continua) 8. La variabilidad en los parámetros de tratamiento entre los estudios dificulta la comparación y ha contribuido al escepticismo respecto a la eficacia 16. Por lo tanto, la optimización de R / NIR-LT está claramente necesario, con los sistemas de cultivo de células capaces de proporcionar el mecanismo de selección de alto rendimiento necesario comparar las múltiples variables. Sin embargo, hay pocos sistemas de iluminación disponibles en el mercado que pueden proporcionar la flexibilidad y el control suficiente sobre wavelength e intensidad para realizar este tipo de experimentos de optimización. Comercialmente dispositivos LED disponibles son generalmente no capaz de entregar múltiples longitudes de onda o intensidades, resultando en investigadores que emplean múltiples dispositivos LED de diferentes fabricantes, que pueden variar no sólo en la intensidad, sino también el espectro de longitud de onda de la luz emitida. Hemos abordado este problema mediante el empleo de una banda ancha de xenón fuente de luz filtrada a través de filtros de interferencia de banda estrecha, generando de ese modo una gama de longitudes de onda y fluencias de luz, lo que permite un estrecho control y precisa de los parámetros de R / NIR-LT. Es importante señalar que la dosis terapéutica de tratamiento se define por el número de fotones que interactúan con el photoacceptor (cromóforo), que, en el caso de R / NIR-LT se postula que el citocromo c oxidasa (COX) 18. La energía fotónica entregado varía con la longitud de onda; es decir, dosis iguales de energía a diferentes longitudes de onda será compreciada de diferentes números de fotones. Por lo tanto, la luz emitida desde el dispositivo fue calibrado para emitir un número igual de fotones para cada una de las longitudes de onda elegidas para ser probado. Hemos desarrollado un sistema que puede ser utilizado para entregar R / NIR-LT en un rango de longitudes de onda e intensidades a las células in vitro y ha demostrado la capacidad de medir los efectos de la entrega R / NIR-LT sobre la producción de ROS en células sometidas a estrés glutamato.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

/ Terapia Red de infrarrojo cercano luz (R / NIR-LT), entregado por láser o diodo emisor de luz (LED), mejora los resultados funcionales y morfológicos en una gama de lesiones del sistema nervioso central in vivo, posiblemente mediante la reducción del estrés oxidativo. Sin embargo, se han mostrado efectos de R / NIR-LT sobre el estrés oxidativo a variar dependiendo de la longitud de onda o la intensidad de la irradiación. Los estudios que compararon los parámetros de tratamiento se carece, debido a la ausencia de dispositivos disponibles en el mercado que ofrecen múltiples longitudes de onda o intensidades, apto para alta rendimiento de procesamiento en los estudios de optimización vitro. Este protocolo describe una técnica para la entrega de la luz en una gama de longitudes de onda e intensidades para optimizar las dosis terapéuticas requeridas para un modelo de lesión dado. La hipótesis de que un método de entrega de la luz, en el que los parámetros de longitud de onda e intensidad podría modificarse fácilmente, podría facilitar la determinación de una dosis óptima de R / NIR-LT para la reducción de especies reactivas de oxígeno(ROS) in vitro. Luz no coherente Xenon se filtró a través de filtros de interferencia de banda estrecha para proporcionar longitudes de onda diferentes (longitudes de onda centrales de 440, 550, 670 y 810 nm) y flujos de energía (8,5 x 10 -3 a 3,8 x 10 -1 J / cm 2) de la luz a las células cultivadas. La salida de luz del aparato se calibró para emitir dosis cuantales terapéuticamente relevantes, iguales de luz en cada longitud de onda. Se detectaron especies reactivas en glutamato destacó las células tratadas con la luz, utilizando DCFH-DA y H 2 O 2 tintes fluorescentes sensibles. Nos éxito librados luz en una gama de longitudes de onda fisiológica y terapéuticamente e intensidades pertinentes, a las células cultivadas expuestas a glutamato como un modelo de lesión del SNC. Mientras que las fluencias de R / NIR-LT utilizados en el estudio actual no ejercer un efecto sobre ROS generado por las células cultivadas, el método de suministro de luz es aplicable a otros Systems incluyendo mitocondrias o más fisiológicamente modelos de cultivo rebanada organotípicos pertinentes aislado, y podría utilizarse para evaluar los efectos sobre una serie de medidas de resultado del metabolismo oxidativo.

La salida de la luz entregada a una longitud de onda de 670 nm fue calibrado usando filtros de densidad neutra con el fin de irradiar las células con una gama de fluencias que abarcan una dosis de luz de 670 nm previamente demostrado ser beneficioso en vivo (0,3 J / cm 2) 20. Como el número de filtros de densidad neutra en frente de la fuente de luz aumenta, la intensidad (W / m 2) disminuyó, lo que permite menos luz para pasar a la zona objetivo. La Tabla 1 presenta los datos de calibr...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método para la Evaluación de los efectos de un rango de longitudes de onda e intensidades de / Terapia de luz del infrarrojo cercano Red sobre el estrés oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

method-for-assessment-effects-range-wavelengths-intensities-rednear

Research

JoVE Journal

Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

8.7K vistas.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Video: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

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 ...

Investigación

Biología

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titulación bioquímica de glucógeno<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análisis de estrés oxidativo en embriones de pez cebra

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La cuantificación de la adhesividad de la célula endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Brecha ocasiones intercelular de comunicación: un biomarcador funcional para evaluar los efectos adversos de las sustancias tóxicas y toxinas, y de Salud Beneficios de Productos Naturales

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Un método para evaluar los efectos de bacteriocinas en la microbiota intestinal de ratones

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Rendimiento medio de selección de ensayos para la evaluación de efectos sobre la Ca2 +-señalización y reacción acrosómica en espermatozoides humanos

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...