Este estudio fue apoyado por una de Ciencias Naturales e Ingeniería del Consejo Canadiense de la subvención (NSERC) de investigación y una Agricultura de Alberta financiación del Consorcio otorga a DRB. AMR está apoyado por una beca NSERC Vanier canadiense de Posgrado, de la Universidad de Alberta ayudantía de enseñanza y la reina Isabel II de becas de postgrado.

Este procedimiento se puede realizar en tubos de 500 l con silicona o 6 mL de poliestireno de fondo redondo de tubos de FACS. Este último se utiliza normalmente en la realización de análisis de viabilidad en relación con otros procedimientos. Todos los volúmenes indicados se refieren a 6 mL de poliestireno de fondo redondo de tubos FACS. De 500 tubos de silicona l, reducir todos los volúmenes de 1 / 5. La concentración óptima para el análisis de citometría de flujo es de 2-4 x 10 6 células por cada 200 l de volumen. La pérdida de células puede ser consecuencia de este procedimiento y por lo tanto se recomienda que cada muestra constará de 4 x 10 6 células en el inicio del procedimiento. 1. Preparación de células <ol><li> Las células de la cosecha - se refieren a procedimientos específicos para las líneas celulares correspondientes o aislamientos de células primarias. </li><li> Centrifugar las muestras a 335 x g durante 10 minutos y decantar el sobrenadante. </li><li> Resuspender las células en 2 ml x 1-fosfato (PBS) - / - (no el calcio, el magnesio no). </li><li> Centrifugar las muestras a 335 x g durante 10 minutos y decantar el sobrenadante. </li><li> Resuspender las células en 1 ml de un tampón de anexina V x vinculante. </li><li> Centrifugar las muestras a 335 x g durante 10 minutos y decantar el sobrenadante. </li><li> Resuspender las células en un buffer de 100 L x anexina V vinculante. </li></ol> 2. Aplicación de anexina V / PI manchas <ol><li> Añadir anexina V de acuerdo con las recomendaciones del fabricante [por ejemplo, 5 l anexina V Alexa Fluor 488 (Molecular Probes, A13201)]. </li><li> Incubar los tubos en la oscuridad durante 15 minutos a temperatura ambiente. </li><li> Añadir 100 ml de un buffer x anexina V la unión a cada tubo de reacción. No debe ser de aproximadamente 200 l en cada tubo. </li><li> Agregar 4 l de PI (Sigma, Cat # P-4864-10 ml) que se ha diluido 1:10 en un tampón x anexina V vinculante (es decir, 1 l PI con 9 l 1 x anexina V buffer de unión). Esto dará lugar a una concentración final de PI 2 mg / mL en cada muestra. </li><li> Incubar los tubos en la oscuridad durante 15 minutos a temperatura ambiente. </li><li> Añadir 500 l 1 x anexina V buffer vinculante para lavar las células. </li><li> Centrifugar las muestras a 335 x g durante 10 minutos y decantar el sobrenadante. </li><li> Resuspender las células en 500 l 1 x tampón de anexina V vinculante y 500 l de formaldehído al 2% para crear una solución de formaldehído al 1% (fijador). Mezcle los tubos de revoloteo suave. </li><li> Fijar las muestras en hielo durante 10 minutos. Por otra parte, las muestras pueden ser almacenadas durante la noche a 4 ° C en la oscuridad. Si el último método que se elija, asegúrese de ser coherente en todos los tubos etiquetados con Anexina V / PI (incluidos los controles de compensación). </li><li> Añadir 1 mL 1 x PBS - / - para cada muestra y mezclar suavemente accionando. </li><li> Tubos de centrífuga a 425 x g durante ocho minutos y decantar el sobrenadante. </li><li> Repita los pasos 2.10 y 2.11. </li><li> Resuspender pellet accionando el tubo. </li><li> Añadir 16 l de 1:100 diluida RNasa (Sigma, R4642) para dar una concentración final de 50 ug / mL. Incubar durante 15 min a 37 ° C. </li><li> Añadir 1 mL 1 x PBS - / - y mezcle suavemente agitando. </li><li> Tubos de centrífuga a 425 x g durante ocho minutos. </li><li> Las muestras están ahora listos para ser analizados. Por otra parte, las muestras se puede utilizar para los pasos de tinción posterior si la anexina / PI tinción se realiza en paralelo con otros procedimientos. </li></ol> 3. Los resultados representativos: Ejemplos de tipos de células y líneas celulares con diferentes grados de tinción PI falsos positivos se muestran en la Figura 1. Para dar cuenta de los eventos de falsos positivos, las células fueron fijadas y luego tratados con RNasa A. Esto se traduce en una disminución significativa en el número de falsos positivos acontecimientos, en comparación con células que no se sometieron a tratamiento RNasa (Figura 2B). La Figura 2c muestra imágenes representativas de las células que se sometieron a RNasa tratamiento, en comparación con células que no recibieron tratamiento RNasa. La Figura 3 muestra cómo la modificación Anexina V / PI protocolo, con la fijación y los pasos RNasa tratamiento, mejora en los protocolos convencionales, al limitar el número de eventos de tinción de falsos positivos. <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="Figura 1" /> Figura 1. Convencionales de propidio protocolos de tinción con yoduro de conducir a un significativo número de falsos eventos positivos. Hemos evaluado previamente el alcance de la tinción de falsos positivos en las células primarias a través de una amplia gama de modelos animales, así como en una variedad de 10 líneas celulares. Representante imágenes muestran el grado de tinción de falsos positivos en tres poblaciones de células únicas (una línea común de células - los macrófagos RAW y dos pilas - murino macrófagos de médula ósea (BMM) y los macrófagos peces de colores primarios de riñón (PKM)). Verdadera acontecimientos positivos mostrar la espera co-localización entre el PI y DRAQ5 dentro del compartimiento nuclear (áreas amarillas representan colocalización entre PI y DRAQ5). DRAQ5 es muy membrane permeable tinte que con precisión las manchas compartimiento nuclear, tanto en las células vivas y muertas, 10,11,12. Para facilitar la determinación visual de la colocalización DRAQ5 mancha se le dio un pseudocolor verde. <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="Figura 2" /> Figura 2. Mejorar la exactitud de la tinción PI nuclear. (A) Se evaluó la exactitud de la tinción PI nuclear basada en el grado de similitud de una serie de bien caracterizado manchas nuclear (BrdU, 7-AAD y DAPI) para DRAQ5. BrdU es un nucleósido sintético que se incorpora en el ADN de las células proliferantes, y como tal, sólo se mancha en el compartimento nuclear. 7-AAD tiene una gran afinidad por el ADN y, de forma similar a la PI, no puede cruzar una membrana plasmática intacta. DAPI también tiene una fuerte afinidad por el ADN nuclear y es el más comúnmente utilizado en la tinción de microscopía de fluorescencia. El grado de similitud era&gt; 99% entre BrdU, 7-AAD, DAPI y DRAQ5, destacando su capacidad de tinción con precisión el núcleo de la célula en los macrófagos RAW 264.7. Por el contrario, la tinción PI citoplasmática basado en protocolos convencionales conduce a una marcada disminución en la similitud de las manchas por ciento en relación con DRAQ5. (B) Resultados similares se observaron en dos pilas a prueba: los macrófagos de médula ósea murina (BMM) y los macrófagos peces de colores primarios de riñón (PKM). Incorporación de 50 mg / ml RNasa A en la etapa específica dentro del procedimiento de tinción elimina no nucleares tinción PI y aumenta significativamente el grado de similitud en relación con DRAQ5 manchas. (C) imágenes representativas de los macrófagos renal primaria muestran el grado de similitud de las células con y sin tratamiento RNasa. Para facilitar la determinación visual de las diferencias entre los tratamientos, DRAQ5 se le dio un color verde. Zonas amarillas representan colocalización entre BrdU y DRAQ5 y PI y DRAQ5. <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="Figura 3" /> Figura 3. Modificado Anexina V / PI reduce significativamente la apoptosis falso / eventos necrótico. Macrófagos primarios de riñón peces de colores (PKM) se tiñeron con convencionales y modificados Anexina V / PI protocolos. Diagramas de dispersión muestran la anexina V / PI mancha en PKM peces de colores. El diagrama de dispersión de la izquierda muestra convencional Anexina V / PI tinción (sin RNasa) de las células de PKM. El diagrama de dispersión de la derecha muestra las células PKM manchadas con la anexina V modificado / protocolo IP (con RNasa). La adición de los resultados de RNasa en una marcada reducción de la tinción PI debido a la eliminación de falsos positivos manchas. PKM células fueron analizadas sobre la base de distintas poblaciones dentro de los progenitores y principios de las culturas (EP), monocitos (Mono) y macrófagos maduros (Mat mac). La reducción en el número de eventos PI positiva, debido a la eliminación de falsos eventos positivos es más pronunciada en las grandes macrófagos maduros que en las pequeñas células progenitoras tempranas. Las conclusiones basadas en protocolos convencionales que erróneamente han sugerido una correlación positiva en la muerte celular de subconjuntos de macrófagos más maduro.

<ol>
	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li><li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li><li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li><li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li><li>Njoh, K. L., Patterson, L. H., Zloh, M., Wiltshire, M., Fisher, J., Chappell, S., Ameer-Beg, S., Bai, Y., Matthews, D., Errington, R. J., Smith, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+analysis+of+the+DNA+targeting+bisalkylaminoanthraquinone+DRAQ5+in+intact+living+cells.">Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.</a> Cytometry Part A. 69, 805-814 (2006).</li></ol>

La anexina V / PI protocolo que aquí se presenta es una versión modificada de los protocolos convencionales y tiene en cuenta la presencia de ARN en el citoplasma, que también tiene gran afinidad por el PI. Introducción de RNasa A (50 mg / ml) después de un paso de formaldehído fijación de un 1% al final del procedimiento de tinción mejora significativamente la precisión de las armas nucleares tinción PI. No se observaron efectos negativos se observan en la tinción nuclear PI o tinción de la membrana plasmática anexina V. Sin RNasa A de tratamiento, hasta un 40% de los resultados de PI mancha puede dar lugar a falsos eventos positivos, lo que conduce a un impacto potencialmente significativo y negativo en las conclusiones posteriores 10. Nuestra modificado Anexina V / PI protocolo de tinción es simple y se ha utilizado con eficacia en una amplia gama de tipos de células (células primarias: murino macrófagos de médula ósea y esplenocitos; porcina células residentes del pulmón, los macrófagos alveolares de pulmón, los ganglios linfáticos mesentéricos aislados, leucocitos de sangre periférica , los esplenocitos, células de pollo blastodermo; macrófagos peces de colores primarios de riñón, la carpa leucocitos en sangre periférica, líneas celulares: macrófagos RAW 264.7, las células Jurkat T, macrófagos porcinos 3D4/31, CCL-71 fibroblastos aleta peces de colores, 3B11 células B bagre 10). Es importante tener en cuenta que las células son diferencialmente afectados por la tinción PI falsos positivos, con pilas por lo general tienen un mayor número de falsos eventos positivos 10. Las diferencias falsas tinción PI positiva entre estas poblaciones celulares puede ser atribuida en parte a diferencias en el tamaño de la celda, pero también puede deberse a diferencias en el contenido de ARN. Como tal incorporación, de este procedimiento es particularmente relevante para los sistemas experimentales que utilizan, entre otras, las células sometidas a estrés genotóxico, las células tratadas con los fármacos detención del ciclo celular como la timidina o hidroxiurea, las células infectadas por virus, y estudios sobre las células embrionarias en la progresión del desarrollo se caracteriza por cambios discretos en la síntesis del ARN celular.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
<tr>
<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
<tr>
<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
<td>Dilute 1:60 in 1 x PBS-/-</td>
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modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

Los estudios de apoptosis celular se han visto significativamente afectados desde la introducción de la citometría de flujo basado en los métodos. Yoduro de propidio (PI) es ampliamente utilizado en conjunción con la anexina V para determinar si las células son viables, apoptosis o necrosis a través de diferencias en la integridad de la membrana de plasma y 1,2 permeabilidad. La anexina V / PI protocolo es un método comúnmente utilizado para el estudio de las células apoptóticas 3. PI se utiliza con más frecuencia que otras manchas nuclear porque es económico, estable y un buen indicador de la viabilidad celular, basado en su capacidad para excluir de colorante en las células vivas 4,5. La capacidad de PI para entrar en una célula depende de la permeabilidad de la membrana; PI no mancha las células apoptóticas en vivo o temprano debido a la presencia de una membrana plasmática intacta 1,2,6. A finales de células apoptóticas y necróticas, la integridad de la membrana plasmática y nuclear disminuye 7,8, lo que permite pasar a través de PI de las membranas, se intercalan en los ácidos nucleicos, y la pantalla de color rojo fluorescente 1,2,9. Por desgracia, nos encontramos con que convencionales anexina V / IP protocolos de conducir a un significativo número de falsos eventos positivos (hasta un 40%), que se asocian con la tinción PI de ARN en el compartimento citoplásmico 10. Las células primarias y líneas celulares en una amplia gama de modelos animales se ven afectados, de células grandes (nuclear: relación núcleo: citoplasma &lt;0,5) que muestra la mayor incidencia 10. En esto, se demuestra una modificación de anexina V / PI método que proporciona una mejora significativa para la evaluación de la muerte celular en comparación con los métodos convencionales. Este protocolo se aprovecha de los cambios en la permeabilidad celular en células de fijación para promover la entrada de la RNasa A en las células después de la tinción. Tanto el momento y la concentración de RNasa A se han optimizado para la extracción de ARN citoplásmico. El resultado es una mejora significativa con respecto convencionales Anexina V / PI protocolos (&lt;5% de los eventos con tinción citoplasmática PI).

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Un método preciso para la evaluación de la muerte celular se describe. El protocolo de mejora sobre convencional anexina V / yoduro de propidio (PI) protocolos, que muestran hasta falsos positivos del 40% los eventos en las líneas celulares y células primarias de una amplia gama de modelos animales.

Modificado anexina V / yoduro de propidio ensayo Apoptosis una evaluación precisa de la muerte celular

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

102.4K vistas.  University of Alberta. Un método preciso para la evaluación de la muerte celular se describe. El protocolo de mejora sobre convencional anexina V / yoduro de propidio (PI) protocolos, que muestran hasta falsos positivos del 40% los eventos en las líneas celulares y células primarias de una amplia gama de modelos animales.

Video: Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificado madre embrionarias de ratón basado en células para la cuantificación de la eficiencia de inducción cardiogénico

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Investigación

Biología

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

A β-glucuronidasa (GUS), basada en células de ensayo Muerte

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

La activación de la apoptosis mediante microinyección citoplasmática del citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

El uso de LysoTracker para detectar la muerte celular programada en embriones y la diferenciación de células madre embrionarias

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

El tomate / GFP-FLP / FRT Método para imágenes en vivo del mosaico adultos<em&gt; Drosophila</em&gt; Las células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Estrategias para Anastasis Seguimiento, una célula de supervivencia Fenómeno que Invierte Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Evaluación del selectivo ARNm traducción en células de mamífero por polisoma perfiles

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Un ensayo de comunicación Unión intercelular yoduro amarillo fluorescente proteína-Gap

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Ensayo único de cultivo de Myofiber para la evaluación de la funcionalidad de las células madre musculares adultas ex vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

Extensión de la vida útil de los neutrófilos con CLON-G y un ensayo de muerte espontánea in vitro

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...