Este trabajo fue apoyado por subvenciones del NIH R37 AI34867 y R01 GM064625 a NWA Agradecemos al Dr. R. Tweten de la Universidad de Oklahoma para el plásmido de expresión de SLO.

1. Transcripcional silenciamiento de la ASM <ol><li> Semilla de 1,5 x 10 5 células HeLa en 2 ml de medio de crecimiento DMEM (DMEM alto en glucosa con 10% de suero bovino fetal (FBS), 2 mM de L-glutamina, 1% de Penn-Strep) en platos inferior 35 mm de vidrio (MatTek) y incubar durante la noche a 37 ° C en el 5% de CO 2 incubadora. </li><li> Al día siguiente, aspirado fuera de los medios de crecimiento y reemplazarlo con 2 ml de DMEM medio de suero reducido (DMEM con FBS al 4%), al menos 1 hora antes de añadir la mezcla de transfección de ARNsi. </li><li> Prepare 4 tubos para la mezcla de transfección siRNA oligo. En los tubos A y C, añadir 250 l de Optimem reducida suero y 4 l de Lipofectamine RNAiMax, por 35 mm de plato a ser transfectadas. En el tubo B, añadir 250 l de Optimem reducen el suero y 8 l (160 pmoles) de control de contenido GC medio oligo (Control siRNA), por placa de 35 mm a transfectar. En el tubo D, añadir 250 l de Optimem reducen el suero y 8 l (160 pmoles) de SMPD1 oligo (siasmo), por placa de 35 mm to ser transfectadas. Incubar los tubos a temperatura ambiente durante 5 min. </li><li> Combinar los tubos A y B para formar el complejo de transfección de ARNsi de control, y los tubos C y D para el ASM ARNsi. Se incuban las reacciones a temperatura ambiente durante 20 min. </li><li> Añadir las mezclas de reacción de transfección siRNA control y ASM siRNA lentamente, gota a gota, en los platos de fondo de 35 mm de vidrio respectivas y se incuba a 37 ° C / 5% CO 2. A las 24 h después de la transfección, aspirar suavemente de los medios de comunicación y reemplazar con medio de cultivo fresco DMEM. Para knockdown ASM eficiente, los platos deben ser incubadas a 37 ° C / 5% CO 2 durante aproximadamente 31 horas (total 55 horas) antes de imágenes en directo. </li></ol> 2. Preparación de los reactivos para Live Microscopía <ol><li> Preparar una solución de toxina de formación de poros recombinante estreptolisina O (SLO) a una concentración de 100 ng / l en frío 1x PBS sin Ca2 + y Mg2 +. La SLO utilizado en estos ensayos es un constitutively mutantes cisteína activa que no requiere agentes para reducir la actividad, proporcionado amablemente por Dr. Rod Tweten de la Universidad de Oklahoma. La toxina se expresó en E. coli BL21 DE3 y se purificó en condiciones nativas, como se ha descrito previamente 16. </li><li> Preparar la solución de stock FM1-43: Resuspender FM1-43 polvo liofilizado en DMSO para crear una solución 25 mM. </li><li> Preparar Solución A: Se diluye la solución madre FM1-43 en DMEM frío con Ca 2 + a una concentración final de 4 mM (se necesitan 1,5 ml por plato de MatTek) y almacenar en hielo hasta que se necesite. </li><li> Preparar la solución B: Diluir la solución madre FM1-43 en DMEM frío sin Ca mM de EGTA 2 + y 10 (Ca 2 +-DMEM libre) a una concentración final de 4 M (1,5 ml necesarios por plato MatTek) y almacenar en hielo hasta necesaria. </li><li> Pre-caliente (37 ° C) alícuotas de 1 ml de Solución A y Solución B en tubos Eppendorf. </li><li> Mantenga las soluciones de hielo de DMEM con Ca 2 +, y Cun DMEM 2 + libre. </li></ol> 3. Imágenes de células vivas de FM1-43 La afluencia en SLO células tratadas y no tratadas <ol><li> Llenar un tamaño medio contenedor de metal poco profunda con hielo picado, invertir en un recipiente de vidrio grande con hielo, y añadir hielo adicional dejando sólo la superficie del fondo del recipiente metálico expuesto. Coloque una toalla de papel húmeda en la parte inferior de metal expuesto para permitir la transferencia térmica uniforme. </li><li> Tome un plato MatTek tratados con siRNA control (muestra 1) y colóquela sobre la toalla de papel húmeda y fría. Se aspira suavemente fuera todos los medios y lavar 3 veces con frío Ca 2 + libre de DMEM. Después del último lavado, aspirado de todos los medios de comunicación en el plato. </li><li> Para la imagen asociada a la célula FM1-43 en las células no herida con SLO (Muestra 1) en la presencia de Ca 2 +, añadir 180 l de solución fría B al cubreobjetos de vidrio centro de la placa inferior de 35 mm de vidrio y se incuba durante 5 min en hielo (Tabla 1). </li&gt; <li> Coloque el plato MatTek en el escenario de un microscopio confocal calentado a 37 ° C y equipado con una cámara ambiental (que mantiene la temperatura, humedad y niveles de CO 2). Encontrar el campo de las células que serán fotografiadas mirando a través de un 40X NA 1.3 objetiva (Nikon). Si está disponible, active PerfectFocus o un dispositivo similar para corregir la deriva térmica. </li><li> Añadir 1 ml de solución A precalentado suavemente a un lado de la placa de 35-mm y reemplace la tapa de la cámara ambiental (Tabla 1). </li><li> Adquirir imágenes de 4 min a 1 fotograma cada 3 s utilizando el software de imagen apropiada (Volocity en el sistema de hilatura de microscopía confocal de disco UltraView Vox Perkin Elmer). La excitación de FM1-43 se lleva a cabo con una línea de láser de 488 nm utilizando un filtro dicroico con un paso de banda de 488 nm, y la discriminación de emisiones se logra a través del uso de un filtro 527 (W55) de emisión. Niveles de potencia láser y sensibilidad de la cámara se ajustan para lograr la exposición de imagen de 100 ms. </li><li> Repita los pasos 3.2 a 3.6 para detectar FM1-43 en las células tratadas con siRNA control sin herir SLO (muestra 2) en ausencia de Ca 2 +, excepto que en el paso 3.5 Solución B debe añadirse (Tabla 1). </li><li> Para medir FM1-43 afluencia en las células tratadas con ARNsi de control heridos con SLO ya sea en la presencia (muestra 3) o ausencia de Ca 2 + (Muestra 4), ​​añadir 180 l de solución fría de SLO B que contiene al cubreobjetos de vidrio centro de la 35 mm de plato con fondo de cristal y se incuba durante 5 min en hielo como en el paso 3.3. Repetir los pasos 3.2 a 3.6; ajustar la adición de cualquiera de las soluciones A o la solución B en el paso 3.5 (Tabla 1). </li><li> Para determinar el papel de la ASM en reparación de la membrana de plasma, repita los pasos 3.2 a 3.6 usando células siRNA tratados con ASM para todas las condiciones (Muestras 5-8) (Tabla 1). </li><li> Para determinar el papel de la esfingomielinasa recombinante extracelular (SM) en lacinética de la reparación de la membrana plasmática (reflejados por la inhibición de FM1-43 afluencia) (muestras 9-10), se añade SM (0-50 mU) a cualquier solución A o la solución B en el paso 3.5 y luego añadieron a las células durante la células vivas formación de imágenes en un volumen total de 1 ml (Tabla 1). </li></ol> 4. Cuantificación de FM1-43 Afluencia en células <ol><li> Después de las películas han sido adquiridos, medir la intensidad de la fluorescencia intracelular de FM1-43 utilizando el software de análisis de imágenes (Volocity Suite, PerkinElmer). Dibujar una región de interés en la región citosólica de cada celda en el campo y aplicar las herramientas de software para determinar la intensidad de fluorescencia FM1-43 media a lo largo de todos los fotogramas de vídeo. </li><li> Para ajustar las variaciones en el FM1-43 tinción inicial, los datos de cada vídeo se expresan como aumento veces en la intensidad de la fluorescencia con el tiempo, y se representaron gráficamente para cada condición diferente. El aumento de veces en la fluorescencia de cada punto de tiempo individual porde células se calcula como F V / F 0, donde F T es la fluorescencia media en el punto de tiempo específico, y F 0 es la fluorescencia media en el momento t = 0 (figuras 1-2). </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Los primeros estudios sobre el daño y la membrana plasmática de reparación mecánica se basó en diversos mecanismos de las células hiriendo, que van desde dejar caer bolas de cristal, micropipeteo, esquila, rayaduras o arañazos. La lectura de todos estos ensayos fue de tipo cualitativo más que cuantitativo, y no dio información precisa sobre la cinética del mecanismo de reparación. La capacidad de las células para volver a sellar su membrana plasmática después de estas formas de lesión se midió por la pre...

Se ha sabido durante varias décadas que reparación de la membrana de plasma después de la lesión mecánica es un 1,2 Ca 2 +-dependiente proceso. Estudios posteriores mostraron que el Ca 2 + afluencia a través de la herida desencadena un proceso vigoroso de la exocitosis de las vesículas intracelulares en el sitio de la lesión, que se requiere para volver a sellar 3,4. Se utilizó la tasa de pérdida de un colorante fluorescente cargado en el citoplasma de las células para evaluar la velocidad de la reparación, y llegó a la conclusión de que el resellado se completó dentro de &lt;30 seg después de la lesión 5. Dos modelos fueron propuestos inicialmente para explicar la necesidad de la exocitosis en la reparación de la membrana plasmática: 1) el modelo de &quot;parche&quot;, lo que sugiere que el Ca 2 + afluencia través de la lesión desencadena la fusión inicialmente homotípica de vesículas intracelulares, formando un &quot;parche&quot; de gran tamaño que haría a continuación, ser aplicada a la membrana plasmática para volver a sellar la herida 6 y 2) el modelo de reducción de la tensión, que propuso que la membrana Added por Ca 2 +-dependiente de la exocitosis en las proximidades de la herida reduciría de plasma tensión de la membrana, facilitando de resellado de la bicapa de 7. El papel de la Ca 2 +-desencadenó la exocitosis en la reparación de la membrana plasmática se ve reforzada por estudios que muestran que los lisosomas contienen una molécula de Ca 2 + sensor, sinaptotagmina VII, lo que facilita su exocitosis y reparación de la membrana plasmática de las células lesionadas 8,9,10,11 . Sin embargo, la evidencia adicional ha indicado que la exocitosis por sí sola no era suficiente para promover la reparación de la membrana de plasma. Además de lágrimas mecánicas en la membrana plasmática, una forma frecuente de lesión de las células es la permeabilización por las toxinas formadoras de poros producidos por bacterias o células inmunes 12,13 14,15. A diferencia de las lágrimas mecánicas, proteínas formadoras de poros se insertan en la membrana plasmática que forma un poro estable, proteína forrado de que no se puede volver a cerrarse simplemente mediante la aplicación de un &quot;parche&quot; de la membrana o por reduccióning tensión de la membrana. Curiosamente, los estudios revelaron que las células de mamífero tienen un mecanismo eficiente para reparar su membrana plasmática después de la permeabilización con proteínas formadoras de poros, y este proceso también requiere la presencia de Ca2 + extracelular 12. Este hallazgo plantea la cuestión de si la supresión de poro transmembrana de la superficie celular era también un proceso rápido, como se observa con heridas mecánicas 5. Sorprendentemente, nuestros estudios recientes revelaron que el resellado de las células permeabilizadas con toxinas formadoras de poros tiene propiedades muy similares a la reparación de heridas mecánicas: el proceso requiere extracelular de Ca 2 +, y se completa dentro de ~ 30 seg. La investigación de este proceso con más detalle, recientemente hemos aprendido que, además de Ca 2 +-exocitosis regulada de los lisosomas, reparación de la membrana plasmática implica una forma rápida de la endocitosis, que se activa por la liberación de la enzima lisosomal esfingomielinasa ácida (ASM) y es esencial no sólo para the eliminación de poros transmembrana, sino también para la reparación de heridas mecánicas 16. Para determinar la cinética de resellado celular después de la permeabilización con proteínas formadoras de poros, en nuestro laboratorio hemos adaptado una metodología de formación de imágenes en vivo que se había utilizado previamente para evaluar el resellado de las fibras musculares aisladas de láser-heridos 17. Este ensayo se basa en las propiedades del colorante lipófilo FM1-43, que se intercala de manera estable en el prospecto exterior de las bicapas de lípidos que aumentan en intensidad de fluorescencia. Cuando la membrana bicapa de plasma se interrumpe ganancias de tinte extracelular acceso a membranas intracelulares, proporcionando un ensayo sensible para detectar lesión de la membrana plasmática y reparar 18,16,19,20. Para adaptar este ensayo para la evaluación de resellado celular después de la permeabilización con proteínas formadoras de poros, que las células pre-incubaron a 4 ° C con la toxina bacteriana estreptolisina O (SLO), que se une al colesterol de la membrana 21. Celular síncronopermeabilización puede entonces lograrse fácilmente moviendo las células de hielo para calentar medio en una platina de microscopio calentado, que activa la oligomerización y cambio en la conformación que conduce a la formación de poros transmembrana. Una ventaja de este enfoque, en los ensayos previamente publicados utilizando heridas láser, es que un mayor número de células puede analizarse simultáneamente en un campo microscópico, proporcionando una mejor toma de muestras de la población celular. Dadas las similitudes mecánicas entre el proceso de resellado de células visto después de la lesión mecánica y la permeabilización con toxinas formadoras de poros, el ensayo como se describe aquí proporciona un método muy versátil y potente para factores implicados en el proceso fundamental de la reparación de la membrana de plasma de disección. Como un ejemplo, se muestra que es posible usar este ensayo para identificar los pasos de Ca 2 + dependientes e independientes del proceso de reparación.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

Lesión de la membrana de plasma es un evento frecuente, y las heridas que ser reparado rápidamente para asegurar la supervivencia celular. La afluencia de Ca 2 + es un evento de señalización clave que desencadena la reparación de heridas mecánicas en la membrana plasmática dentro de ~ 30 seg. Estudios recientes revelaron que las células de mamíferos también vuelven a sellar su membrana plasmática después de la permeabilización con formador de poros toxinas en un proceso de 2 +-dependiente de Ca que implica la exocitosis de la esfingomielinasa ácida enzima lisosomal seguido por endocitosis de poro. Aquí se describe la metodología utilizada para demostrar que el resellado de las células permeabilizadas por la toxina estreptolisina O también es rápida y dependiente de Ca 2 + afluencia. El diseño del ensayo permite la sincronización del evento lesión y una medición cinética precisa de la capacidad de las células para restaurar la integridad de la membrana de plasma mediante formación de imágenes y la cuantificación de la medida por la que el colorante liphophilic FM1-43 llega a las membranas intracelulares. Este vivo als ensayoO permite una evaluación sensible de la capacidad de exógenamente añadido factores solubles tales como la esfingomielinasa para inhibir FM1-43 afluencia, que refleja la capacidad de las células para reparar su membrana plasmática. Este ensayo nos permitió demostrar por primera vez que la esfingomielinasa actúa aguas abajo de Ca 2 +-dependiente de la exocitosis, ya que la adición de la enzima extracelular promueve resellado de las células permeabilizadas en ausencia de Ca 2 +.

Las bajas concentraciones de reparación de la membrana plasmática esfingomielinasa (SM) de rescate de bacterias en las células agotadas en esfingomielinasa ácida lisosomal. Usando el ensayo de formación de imágenes FM1-43, que anteriormente mostró que las células deficientes para el ASM enzima lisosomal y expuestos a SLO hiriendo en la presencia de Ca 2 + tienen un defecto de membrana plasmática es rescatado por adición extracelular de la enzima humana recombinante 19....

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Formación de imágenes en vivo de las células expuestas a la colorante lipófilo FM1-43 permite la determinación precisa de la cinética por el cual las toxinas formadoras de poros son retirados de la membrana plasmática. Este es un ensayo sensible que puede ser utilizado para evaluar las necesidades del Ca 2 +, esfingomielinasa y otros factores en la reparación de la membrana de plasma.

Viva Imaging Ensayo para la evaluación de los Roles de Ca<sup&gt; 2 +</sup&gt; Y esfingomielinasa en la reparación de los poros de la formación de toxina Heridas

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

live-imaging-assay-for-assessing-roles-ca2-sphingomyelinase-repair

Research

JoVE Journal

Biology

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

11.6K vistas.  University of Maryland.  Formación de imágenes en vivo de las células expuestas a la colorante lipófilo FM1-43 permite la determinación precisa de la cinética por el cual las toxinas formadoras de poros son retirados de la membrana plasmática. Este es un ensayo sensible que puede ser utilizado para evaluar las necesidades del Ca 2 +, esfingomielinasa y otros factores en la reparación de la membrana de plasma. 

Video: Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

La preparación de los Drosophila Los embriones para Live-Imaging utilizando el protocolo de gota

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Investigación

Biología

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de Imagen Ca 2 + Señalización en el esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

Resonancia de spin electrónico micro imágenes en vivo de las especies para la asignación de oxígeno

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Imágenes en vivo del calcio celular combinado con el silenciamiento de genes mediada por siRNA Identifica Ca 2 + Canales de fugas en la membrana del ER y sus mecanismos de regulación

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un ensayo de explante para evaluar el comportamiento celular del mesénquima craneal

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Imágenes de células vivas de las primeras autofagia Eventos: Omegasomes y más allá

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...