Los autores desean agradecer al Prof. Nicolás Smirnoff, de la Universidad de Exeter por su consejo y la Universidad de Exeter Bioimagen Fondo. El financiamiento fue proporcionado por la Biotecnología del Reino Unido y Ciencias Biológicas de Investigación (subvención de referencia BB/E002682/1).

El protocolo se indican a continuación se describe un método sencillo de usar PFD como un medio de infiltración de montaje en las hojas de Arabidopsis thaliana, pero anticipamos que este método puede ser utilizado en una variedad de aplicaciones en las imágenes de aire rico en tejidos que se desea. 1. Montaje de muestras de hojas en PFD <ol><li> Preparar un portaobjetos con una junta de gas permeable de polidimetilsiloxano (PDMS, Carolina Gel de observación). PDMS es un polímero viscoelástico y puede ser moldeado para proporcionar una cámara adaptada a las necesidades experimentales. </li><li> Equilibre PFD con el aire. Esto puede lograrse mediante burbujeo de aire o agitando un pequeño volumen de PFD en una botella llena de aire. </li><li> PFD decantar en una placa de Petri abiertas y flotar una planta entera o de hojas eliminado en PFD durante 5 minutos. El tejido debe ser transparente, que recuerda a los tejidos vitrificados (Figura 2 (a)). Las hojas pueden aparecer más oscura o más clara que antes de la exposición a PFD, dependenciadiendo de las condiciones de iluminación y la edad del tejido utilizado. </li><li> Llenar la cámara de PDMS con aire equilibrada PFD y transferir cuidadosamente las muestras de tejido de la placa de Petri de incubación de la cámara de PDMS. Sello de la diapositiva con un cubreobjetos y la imagen de acuerdo a los requerimientos experimentales. </li><li> Nota: PFD también funciona bien en una cámara abierta construido sobre un portaobjetos o en una cámara de perfusión y es compatible con grasa de silicona. PFD no es compatible con los componentes de teflón, ya que las disuelve. </li></ol> 2. Los resultados representativos: Examen microscópico de las muestras de hojas PFD-incubadas muestra que la mayoría de los espacios aéreos están inundadas. Figura 2 (b) muestra los espacios aéreos inundado recombinante GFP suspendido en PFD. Es evidente que la hoja está inundado tras la incubación con PFD, pero que las bolsas de aire ocasional puede permanecer. El agua no se inunde la hoja en esas condiciones. LSCM de las muestras se incuban y se monta en un<html> ir, el agua o muestra PFD PFD que da una ventaja sobre el agua y el aire en la profundidad de imagen posible (Figura 3). El ejemplo de la Figura 3 se muestra Arabidopsis aire, agua y PFD montado deja expresar cytosolically localizada Venus, una variante de YFP 11, que se expresa constitutivamente en el promotor 35S. Podemos ver un aproximado de 2 veces más en profundidad de la imagen cuando se compara PFD y el agua. Cabe señalar, sin embargo, que la mejora precisa ve cuando se utiliza varía según el PFD apertura numérica de la lente y el tipo de tejido utilizado. Hemos visto corriente citoplasmática y el alargamiento de la raíz del cabello PFD plántulas tratadas, todos ellos indicativos de las plantas sanas. Además hemos demostrado que dos Fv / Fm, una medida del funcionamiento fotosintético de las plantas 12 permanece dentro de límites tolerables en escalas de tiempo utilizadas en las imágenes. 3394fig1.jpg &quot;/&gt; Figura 1. Planta de la complejidad y las implicaciones de la hoja óptica Representación esquemática que muestra las características anatómicas de la A. thaliana hojas en relación con la óptica puesta en marcha. Abreviaturas utilizadas son obj. = Lente objetivo, imm. = Líquido de inmersión, cov. = Cubreobjetos, mnt. = Medio de montaje, corte. = Cutícula, ad. ep. = Epidermis adaxial, st. = Poro estomático, sp. = Mesófilo esponjoso, como = el espacio aéreo, amigo. = Empalizada mesófilo, vb = paquete vascular, ad. ep. = Adaxial epidermis. Las paredes celulares están indicados por las líneas de color negro. <img alt="Figura 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Figura 2. PFD penetra fácilmente hojas de Arabidopsis (A) Las hojas se incubaron en el aire, el agua o PFD por 5 minutos y la imagen utilizando una Leica DCF3000FX cámara digital con un microscopio Leica MZ16F (Leica Microsystems (Reino Unido) Ltd., Milton Keynes, Reino Unido). El logotipo JoVe fue impreso en la película de acetato e iluminado lado a otrom por debajo con una caja de luz. Tiempo de exposición fue de 89 ms y todas las imágenes fueron recogidos y tratados de forma idéntica. Las barras de escala representan el 2 mm. (B) llevar a PFD recombinante purificada la proteína verde fluorescente (GFP) delimita los espacios aéreos en vivo. GFP es de color verde falsa, y el rojo se utiliza para mostrar auto-fluorescencia de clorofila, que delimita las células del mesófilo. (Figura 2 (b) que se reproducen con el permiso y todos los detalles técnicos disponibles en Littlejohn et al. 2010 2). Las barras de escala representan 25 m. <img alt="Figura 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Figura 3. PFD mejora de imagen confocal en las hojas LSCM imágenes que muestran citoplasma localizada Venus fluorescencia (verde) y la autofluorescencia de clorofila (rojo) en A. thaliana hojas intactas imágenes en el aire, el agua o PFD. Longitud de onda de excitación fue 514 nm y emisión de fluorescencia se registró a 518 nm 604 nm de Venus y en 657 nm679 nm de los cloroplastos. Cada panel es una sola sección confocal tomado de un Z-stack de 11 imágenes confocal adquiridos en intervalos de 5 micras. Estos fueron extraídos de un total Z-stack de 59 imágenes tomadas a intervalos de 1 m, que se han utilizado para producir películas complementarias. Las mediciones de profundidad se dan en relación a la superficie epidérmica. Imágenes representativas, que se procesaron de manera idéntica, se muestran. Los detalles experimentales, tales como ajustes del microscopio son idénticos a los encontrados en Littlejohn et al., 2010 2. Scalebars representan el 20 micras.

<ol>
	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

No hay conflictos de interés declarado.

Esta es una técnica sencilla y fácil de usar para mejorar la microscopía llenas de aire o complejos ópticamente tejidos. Hemos demostrado que la técnica tiene algunas ventajas importantes, y esperamos que se va a utilizar para dilucidar cuestiones biológicas relacionadas con el aire rico en los tejidos. Por ejemplo, sería una elección natural para los estudios de ataque de patógenos en el mesófilo de la planta o en los pulmones. También son conscientes de las limitaciones de la técnica. Estamos trabajando en una mejor adecuación del índice de refracción, el uso de otros modos de microscopia, y la reposición de perfluorocarbono medio de montaje durante la larga escala temporal experimentos. También reconocemos, sin embargo, que una de las principales ventajas de la PFC, es decir, ser biológicamente inerte tiene un reverso. PFC no se disuelven fácilmente las moléculas biológicas que también implica que no pueden ser fácilmente utilizados para entregar los compuestos de interés como las hormonas, las drogas, y otras moléculas pequeñas o iones.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Perfluorodecalina </td><td> F2 productos químicos </td><td> N / A </td><td> Teléfono a la orden. </td></tr><tr><td> Carolina Observación Gel </td><td> Hojas Biológica </td><td> 13-2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

El problema de la adquisición de imágenes de alta resolución de profundidad en las muestras biológicas es ampliamente reconocido 1. En el aire lleno de tejidos, tales como el mesófilo esponjoso de las hojas de la planta o en los pulmones de vertebrados más dificultades surgen de múltiples transiciones en el índice de refracción entre los componentes celulares, entre las células y su espacio aéreo y entre el tejido biológico y el resto del sistema óptico. Por otra parte, los desajustes del índice de refracción conducir a una atenuación de la excitación y la emisión de la señal de fluoróforo en microscopía de fluorescencia. Se describe aquí la aplicación de los perfluorocarbono, perfluorodecalina (PFD), como un medio de imágenes infiltrativas que ópticamente mejora la microscopía confocal de barrido láser (LSCM) muestra imágenes en profundidad, sin necesidad de recurrir a un aumento perjudicial en potencia del láser y tiene un impacto mínimo fisiológico 2. Se describe el protocolo para el uso de PFD con el tejido Arabidopsis thaliana de la hoja, que es ópticamente complejas como resultado de suestructura (figura 1). PFD tiene una serie de atributos que lo hacen adecuado para este uso 3. El índice de refracción de PFD (1.313) es comparable con la del agua (1,333) y se acerca más a la de citosol (aprox. 1,4) que el aire (1.000). Además, el PFD es fácilmente disponible, no fluorescente y no es tóxico. La baja tensión superficial de PFD (19 dinas cm -1) es menor que la del agua (72 dinas cm -1) y también por debajo del límite (25 - 30 cm dina -1) para la penetración de los estomas 4, que permite a las inundaciones los espacios aéreos del mesófilo esponjoso sin la aplicación de un vacío potencialmente destructivos o el agente tensioactivo. Finalmente y lo más importante, PFD tiene una gran capacidad de disolución de CO 2 y O 2, que permite el intercambio de gases se mantengan en el tejido inundadas, minimizando así el impacto fisiológico en la muestra. Estas propiedades han sido utilizadas en diversas aplicaciones que incluyen la respiración líquida parcial y la inflación de pulmónción 5,6, la cirugía 7, 8 sangre artificial, la oxigenación de crecimiento de los medios 9, y los estudios de la formación de cristales de hielo en las plantas 10. En la actualidad, es común para montar los tejidos en el agua o tampón acuoso de imagen confocal en vivo. Consideramos que el uso de PFD como un medio de montaje representa una mejora en la práctica existente y permite la simple preparación de las muestras en vivo de hoja entera de imágenes.

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Se describe el uso de perfluorodecalina como un medio de infiltración de montaje. Este es un método sencillo para mejorar la profundidad de la imagen en<em&gt; Arabidopsis thaliana</em&gt; Tejido foliar, con un mínimo impacto fisiológico.

Un método simple para imágenes Arabidopsis Uso de hojas perfluorodecalina como un medio de imágenes infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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Research

JoVE Journal

Biology

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

21.5K vistas.  The University of Exeter. Se describe el uso de perfluorodecalina como un medio de infiltración de montaje. Este es un método sencillo para mejorar la profundidad de la imagen en Arabidopsis thaliana Tejido foliar, con un mínimo impacto fisiológico.

Video: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Investigación

Biología

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo</emPollo> Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análisis específico de célula de<em&gt; Arabidopsis</em&gt; Hojas usando citometría de flujo

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

Configuración de un microscopio de luz Hoja simple para<em&gt; En Toto</em&gt; Imágenes de<em&gt; C. elegans</em&gt; Desarrollo

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La técnica de infiltración-centrifugación para extracción de apoplástico fluido procedente de hojas de la planta Uso<em&gt; Phaseolus vulgaris</em&gt; Como ejemplo

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging en hidrogeles tridimensionales

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Discriminación y Caracterización de las Poblaciones Heterocelulares Usando Técnicas de Imagen Cuantitativa

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Método estandarizado para la esterilización de alto rendimiento de semillas de Arabidopsis

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Complementación de luciferase imagen ensayo en hojas de Nicotiana benthamiana para determinar transitoriamente proteínas interacción dinámica

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Proyección de imagen de FITC-dextrano como reportero para exocitosis regulada

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...