We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1. Aequorina Based Ca 2 + Imaging Uso del sistema FAS <ol><li> Preparar plántulas para imágenes de luminiscencia. Esterilizar semillas de plantas de Arabidopsis que expresan Aequorina con una solución de lejía al 10% que contiene 0,01% de Triton-100. Siembre las semillas estériles en un plato cuadrado (10 x 10 cm cuadrados caja de Petri con rejilla,) que contiene toda su fuerza MS (Murashige y Skoog Basal Sal Mezcla), 1% de sacarosa y 1,2% de agar. Colocar las placas verticalmente en una cámara de crecimiento después de la estratificación a 4 ° C durante 2 días (Figura 1A). </li><li> Transferir las plántulas sobre una película. Colocar una película adhesiva (Figura 1B) en la parte superior de 7-10 días de edad plántulas que crecen en la placa. Empuje suavemente la película con la mano para asegurarse de que las plántulas se adhieren a la película (Figura 1C). Pelar la película con cuidado para que las plántulas permanecen adheridos a la película (Figura 1D y 1E) </li><li> Se incuban las plántulas con cofactor. Colocar la unaplántulas dhered sobre la placa cuadrada (10 x 10 cm) que contiene 15 ml de 2 mg / ml h-CTZ (coelenterazina) en agua. Incubar las plantas de semillero a temperatura ambiente durante 4 horas a toda la noche (Figura 1F). </li><li> Preparar para la imagen de luminiscencia. Tome la película fuera de la solución h-CTZ y cortar por la mitad, formando dos piezas. Coloque cada pieza de película con plántulas cara arriba en dos placas diferentes. Deje las placas en la oscuridad durante 5 min. </li><li> Adquirir imágenes de luminiscencia. En la oscuridad, colocar las dos placas junto a la otra en la etapa del sistema de imágenes de luminiscencia (Figura 1G). Adquirir imágenes inmediatamente después de la adición de 20 ml de solución de estímulos a las placas de forma simultánea. </li><li> Analizar imágenes de luminiscencia. Elija el mismo rango de visualización para todas las imágenes de luminiscencia. Recortar la región de interés (ROI) y generar las imágenes como archivos JPEG (Figura 2A). Alternativamente, exportar las imágenes como archivos de formato SPE e importarlos en elImageJ software de análisis de imagen. Medidas de ajuste para el cálculo del valor medio gris del ROI. Seleccione el mismo tamaño del área de retorno de la inversión y medir los datos de grises y presentes medios como gráficos de barras (Figura 2B). </li></ol> 2. vivo Cell Confocal Ca 2 + Imaging <ol><li> Preparar plántulas para la imagen confocal. Esterilizar semillas de plantas de Arabidopsis que expresan case12 con una solución de lejía al 10% que contiene 0,01% de Triton. Siembre las semillas estériles en una placa que contiene la fuerza completa sales MS, sacarosa al 1%, y 1,2% de agar. Coloque las placas verticalmente en una cámara de crecimiento después de la estratificación a 4 ° C durante 2 días. </li><li> Cámaras de imagen de instalación <ol><li> Montar una cámara de imágenes usando un portaobjetos y cubreobjetos (Sala A). Coloque un trozo de lana de algodón empapado de agua en el medio de la corredera. A continuación, traslado de uno o dos 5 días plántulas de la placa en la parte superior del agua empapada lana de algodón. Se adhieren pequeños trozos de arcilla a cada esquina de un cubreobjetos, unad lugar cubreobjetos en la parte superior de las plantas de semillero para crear un espacio (cámara) entre el cubreobjetos y portaobjetos (Figura 3A, panel izquierdo). Conectar un extremo del tubo de polietileno (0,58 mm de diámetro) a una jeringa de 1 ml y colocar el otro extremo del tubo inmediatamente adyacente a la cámara. Sostenga el tubo en su lugar con cinta (Figura 3A, panel derecho). </li><li> Montar una cámara de imágenes utilizando una cámara de plexiglás (Sala B). <ol><li> Extender una capa fina de grasa de silicona alrededor de dos tubos de polietileno (0,58 mm de diámetro) y presione los tubos revestidos en los canales a cada lado de la cámara de plexiglass (Figura 3B panel de la izquierda). Extender una capa fina de grasa de silicona sobre la superficie de la cámara que contiene las ranuras del tubo y presione un cubreobjetos en la grasa para sellar un lado de la abertura de la cámara (cortadas). </li><li> Transferir una antigua planta de semillero cinco días a partir de la placa en la cámara y coloque un trozo de algodón empapado con agua en la parte superior of la plántula. Extienda la grasa de silicona sobre la otra superficie de la cámara, y presione otro cubreobjetos en la parte superior para sellar. Conectar el extremo de uno de los tubos a una jeringa de 1 ml. Deje el extremo del otro tubo (panel de la derecha Figura 3B) abierta. </li></ol></li><li> Preparar una cámara de imágenes utilizando una cubierta de vidrio cámaras (Cámara C). Esterilizar el cristal de la cubierta de cámaras con etanol al 70% y se deja en el capó hasta que se seque. Añadir 0,6 ml o 0,2 ml de fuerza completa medio MS que contenía 1% de sacarosa y 0,5% de fitagel en cada pocillo de una cámara de 2 pocillos o una cámara de 8 pocillos (panel de la derecha Figura 3C), respectivamente. Esterilizar semillas y sembrar las semillas directamente en una cubierta de vidrio cámaras que contiene una fina capa de gel transparente y dejarlos crecer verticalmente durante 5 días (Figura 3C). </li></ol></li><li> Adquirir imágenes utilizando un microscopio confocal. Para aplicar estímulos de estrés, como la sal, el frío o peróxido, inyectar lentamente alrededor de 200 l de NaCl 150 mM, hielo-cold agua o 1 mM H 2 O 2 solución en la cámara (Cámara A o B) justo antes de la adquisición de imágenes, o agregar suavemente 500 l o 100 l de solución de estímulo para el pozo de una cámara de 2 o de 8 pocillos, respectivamente . Capturar imágenes inmediatamente después de aplicar la solución de estímulo utilizando un microscopio confocal Nikon A1R de escaneo láser invertida con un lente de inmersión en agua 20X (apertura numérica 0,75). Reúne una serie temporal de imágenes a intervalos de 4 segundos con longitudes de onda de excitación y emisión de 488 nm y 500-550 nm, respectivamente, y con una resolución de píxel de 512 x 512. </li><li> Análisis de Imagen Usando Nikon Elements, ROIs fueron elaboradas alrededor de cada celda (o área) de interés. Intensidad total dentro de cada ROI se mide en el tiempo utilizando el cuadro de diálogo Medición del tiempo (ImageJ se podría utilizar en su lugar). Mediciones totales de intensidad fueron exportados y tratados en Datagraph. Ca 2 + amplitud pico se define como la intensidad máxima de menos intensidad de descanso, la duración como ªtiempo e entre el inicio y la finalización de un pico, y el período como el intervalo de tiempo entre los picos pico adyacentes de dos picos. t-test fue utilizado para comparar las medias. </li></ol>

<ol>
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The authors have nothing to disclose.

Hemos demostrado un sistema FAS para el registro de la espacio-temporal Ca 2 + respuesta de plántulas de Arabidopsis. Este sistema de grabación FAS 2 + Ca proporciona un enfoque sencillo, sensible y robusto que se podría adaptar para medir la dinámica de Ca 2 + desencadenadas por diversos estímulos, además de los estímulos de estrés abióticos que se presentan aquí. El uso de este sistema, podemos comparar fácilmente tejido o específicos estímulos espaciales-temporales d...

La célula de la planta responde al medio ambiente a través de señalización que coordina las acciones celulares. Un evento de señalización temprana de la célula en respuesta a estímulos del medio ambiente es un aumento transitorio de Ca 2 +. El patrón, o la firma de un aumento transitorio en la concentración de Ca2 + libre se caracteriza por su amplitud, frecuencia, y duración. Firmas Ca 2 + espacio-temporales diferentes regulan diferentes actividades celulares 1. Estímulos específicos, como el calor, el frío, la sal, la sequía, la luz o las hormonas vegetales, pueden afinar la actividad espacio-temporal de Ca 2 + canales de membrana localizada y / o transportistas, lo que resulta en específicos de Ca 2 + firmas. Aunque Ca 2 + transportistas han sido bien caracterizado, se sabe poco sobre las identidades moleculares y las funciones de los canales de Ca 2 + en las plantas 1. Pantallas genéticos para mutantes con alteración de Ca 2 + respuesta al estrés estímulos pueden ser un apr efectivaoach para la identificación de los componentes que componen las firmas de Ca 2 +. Ca 2 + sistemas de detección Recientemente basa varios Aequorina han sido desarrollados para facilitar las pantallas de genética para el Ca 2 + señalización componentes en respuesta al ataque de patógenos y estrés abiótico 2-4. Aequorina fue utilizado por primera vez para detectar Ca 2 + señales en plantas a principios de 1990 5. Desde entonces, la Aequorina se ha dirigido a diferentes compartimentos celulares, tales como el citoplasma 5, el núcleo 6, 7 cloroplastos, tonoplasto 8, 9 mitocondrias, y el estroma 10, así como a diferentes tipos de células en la raíz para supervisar celda específica Ca 2 + señales 11. Mediciones de Ca 2 + basado Aequorina revelan la espacial y temporal Ca 2 + respuesta de una población de células para estresar estímulos. Sin embargo, en la mayoría de los casos, las respuestas de la Ca 2 + de las células individuales son unsynchronizado en el tejido de responder 4. Por lo tanto, la grabación Aequorina Ca 2 + no informa necesariamente la señal de Ca 2 + en las células individuales. En los últimos años, la proteína fluorescente codificada genéticamente (FP) a base de Ca 2 + indicadores, como cameleon amarilla (YCs) 12 y CASEs12 13 se han utilizado para estudiar Ca 2 + señalización con alta resolución subcelular. YCs son la transferencia de energía de resonancia fluorescente (FRET) a base de Ca 2 + indicadores, que contiene PPC y YFP variantes vínculo del Ca 2 + vinculante calmodulina proteína y péptido de unión a calmodulina-M13. La calmodulina sufre un cambio conformacional ya que se une a Ca 2 +, con lo que trae PPC y YFP más cerca juntos, resultando en una mayor transferencia de energía (FRET mejorada). El nivel de FRET con el tiempo, más o menos calculado como la relación de las intensidades de señal de YFP PPC, refleja la dinámica intracelular de Ca 2 +. Varias versiones TA se han utilizado en las plantas. YC3.6 era targeted al citosol 14,15, el núcleo 16, 17 mitocondrias y membrana plasmática 18, y YC4.6 y D4ER fueron atacados a Urgencias 15,19, y D3cpv fue dirigido a los peroxisomas 20. Las plantas transgénicas que expresan YCs permiten la formación de imágenes en vivo de células de Ca 2 + dinámica dentro de los diferentes compartimentos celulares de diferentes tipos de células. Casos (presumiblemente Ca lcium sí Nsor) son proteínas individuales circularmente permutados fluorescentes (CPFPS) albergan una calmodulina y el péptido de unión a calmodulina-M13. Tras la unión a Ca 2 +, CASEs sufren cambios conformacionales, llevando a un aumento de la intensidad de fluorescencia. La correlación entre la respuesta de fluorescencia del CASE y concentración de Ca2 + intracelular de Ca 2 + permite la dinámica que se deben medir cuantitativamente. La variante Case12 ha 12 veces mayor fluorescencia en la Ca 2 + formas saturados. N. plantas benthiminana transitoriamente expresser Case12 o plantas de Arabidopsis que expresan establemente caso 12 se utilizaron para estudiar Ca 2 + señalización en defensa y abióticos de estrés 4,21. Asíncrono espacial y temporal de Ca 2 + oscilaciones en células que responden al ataque de patógenos, o al estrés deshidratación han sido reveladas con basado Case12 Ca 2 + de imágenes. A continuación, presentamos las instrucciones detalladas para Aequorina imágenes luminiscencia basada de tejidos y estímulos específicos de Ca 2 + en la dinámica de plántulas de Arabidopsis, y de imagen confocal de citosólicas y Ca 2 + dinámica nuclear en células de la raíz de Arabidopsis que expresan Caso 12. luminiscencia imágenes de FAS podría ser adaptado para analizar el estrés inducido por la dinámica de Ca2 + en las plantas o tejidos que no se describen aquí intactos, o para cribar poblaciones de plantas de Arabidopsis mutagenizadas para mutantes con el estrés alterada inducida por Ca 2 + señales. La configuración de imágenes de células vivas Ca2 + podría adaptarse para analizar Ca2 + dinámicas dentro de diferentes compartimentos subcelular o en diferentes tipos de células utilizando otros indicadores de Ca 2 +.

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			<td height="21" style="height:21px;">1 mL syringe&#160;</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">53548-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silicone grease&#160;</td>
			<td style="width:172px;">Beckman</td>
			<td style="width:175px;">335148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well chambered cover glass&#160;</td>
			<td>Nalge Nunc international</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">8-well chambered cover glass</td>
			<td>Nalge Nunc international</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Imaging software</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td>Nikon Elements&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">ImageJ</td>
			<td style="width:172px;">NIH</td>
			<td style="width:175px;">N/A</td>
			<td>Public domain, Java-based image processing program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DataGraph</td>
			<td>Visual Data Tools Inc</td>
			<td style="width:175px;">N/A</td>
			<td>DataGraph 3.1.1 is the newest version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coelenterazine</td>
			<td>NanoLight Technolgies</td>
			<td style="width:175px;">#301B NF-BCTZ-FB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">All purpose Bleach</td>
			<td style="width:172px;">Any local store</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Triton X-100</td>
			<td style="width:172px;">Fisher</td>
			<td style="width:175px;">BP151500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">MS salt</td>
			<td style="width:172px;">Phytotechnology Labs</td>
			<td style="width:175px;">M524-50L</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">sucrose</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">BDH8029-12KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">A1296-5KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Phytagel</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">P8169-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spatial and temporal ca2+ signals in arabidopsis plants

Señales de desarrollo y ambientales inducen Ca 2 + fluctuaciones en las células vegetales. Espacio-temporales de Ca 2 +-patrones de estímulo específico son detectados por las proteínas de unión a Ca 2 + celulares que inician Ca 2 + cascadas de señalización. Sin embargo, todavía sabemos poco sobre cómo estímulo señales específicas Ca 2 + se generan. La especificidad de una señal de Ca2 + puede atribuirse a la regulación sofisticada de las actividades de los canales y / o transportadores de Ca 2 + en respuesta a un estímulo dado. Para identificar estos componentes celulares y entender sus funciones, es crucial usar sistemas que permiten una grabación sensible y robusta de señales de Ca 2 +, tanto en el tejido y niveles celulares. Genéticamente codificados Ca 2 + indicadores que están dirigidos a diferentes compartimentos celulares han proporcionado una plataforma para la célula viva imagen confocal de celulares Ca 2 + señales. Aquí se describe la instruccións para el uso de dos sistemas de detección de Ca 2 +: Aequorina basa FAS (adhesivo de película) plántulas luminiscencia Ca 2 + de imágenes y basado case12 de fluorescencia confocal de células vivas Ca 2 + de imágenes. De imágenes de luminiscencia usando el sistema FAS ofrece una detección simple, robusto y sensible de espaciales y temporales Ca 2 + señales a nivel de los tejidos, mientras que la célula viva imagen confocal utilizando Case12 ofrece detección simultánea de citosólicas y nucleares Ca 2 + señales con una resolución alta.

Manitol, NaCl y H 2 O 2 se utilizaron como indicadores de la deshidratación, la sal y el estrés oxidativo estímulos, respectivamente. Para comprobar si el ion de metal pesado Cu 2 + sinergia con cualquiera de estos tres estímulos de estrés, se comparó la Ca 2 + respuesta a cada uno de los estímulos en la presencia o ausencia de Cu 2 +. Como se muestra en la Figura 2, las imágenes de luminiscencia FAS reveló que las plantas de semillero Arab...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

La señalización de Ca2 + regula diversos procesos biológicos en las plantas. Aquí presentamos los enfoques para el control de estrés abiótico inducida espacial y temporal Ca 2 + señales en las células y tejidos que usan las codificados genéticamente Ca 2 + indicadores Aequorina o Case12 Arabidopsis.

Medición Espacial y Temporal Ca<sup&gt; 2 +</sup&gt; Las señales en plantas de Arabidopsis

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K vistas.  Purdue University.  La señalización de Ca2 + regula diversos procesos biológicos en las plantas. Aquí presentamos los enfoques para el control de estrés abiótico inducida espacial y temporal Ca 2 + señales en las células y tejidos que usan las codificados genéticamente Ca 2 + indicadores Aequorina o Case12 Arabidopsis. 

Video: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

Modelado de membranas biológicas con placas de circuitos y medición de señales eléctricas en los axones: Ejercicios de laboratorio para estudiantes

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Investigación

Biología

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Capa de hielo: Un método para el cultivo Arabidopsis Y plantas de tomate en las placas de 96 pocillos de alto rendimiento para genotipado

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Medición de Ca intracelular<sup&gt; 2 +</sup&gt; Cambios en el esperma humano mediante cuatro técnicas: Fluorometría convencional, se detuvo Fluorometría Flow, citometría de flujo y Single imágenes de células

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Análisis automatizado de Dinámica Ca<sup&gt; 2 +</sup&gt; Las señales en secuencias de imágenes

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

En tiempo real en Vivo la grabación de señales de calcio Arabidopsis durante la alimentación de insectos utilizando un Biosensor fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Aplicaciones de la cartografía espacio-temporal y técnicas de análisis de partículas para cuantificar Ca intracelular2 + señalización In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Secuenciación de ARN-Secuenciación de ARN-Captura láser para Análisis de Expresión Genética Espacial y Temporal Específica de Tejidos en Plantas

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Wide-Field, Imágenes en tiempo real de señales de heridas locales y sistémicas en Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Un método de inoculación fácil y flexible para evaluar con precisión los fenotipos de infección por mildiú polvoriento de Arabidopsis y otras plantas

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...