Nos gustaría dar las gracias al CIRM para apoyar este trabajo. La línea celular fue generosamente donada por el Dr. Andras Nagy en el Mount Sinai Hospital, Toronto, Ontario.

1. Hacer Shrinky Dink-Moho <ol><li> Imprimir el modelo deseado en la hoja de Shrinky-dink con una impresora buena definición. </li><li> Hornee Shrinky-dink hoja a 163 ° C durante unos 10 minutos, o hasta que esté completamente reducido y haber adquirido una forma regular. </li><li> Después de Shrinky-dink molde se haya enfriado, se sumergen en un baño de isopropanol hasta que la superficie total es de apenas cubiertos. </li><li> Cuidadosamente, rociar un poco de acetona sobre el molde y agitar contenedores un par de veces. Añadir más isopropanol para vaciar el exceso de acetona y repetir este paso varias veces hasta que Shrinky el molde se ve limpio. </li><li> Molde de sumergir en agua destilada durante 10 minutos para lavar cualquier resto de solvente orgánico. </li><li> Aire limpio Shrinky el molde. Volver a calentar durante unos 5 minutos a 163 ° C. Esto tinta compacta y evaporar cualquier solvente restante. </li></ol> 2. Hacer PDMS micropocillos <ol><li> Prepare una mezcla de agente de 10:01 PDMS / curado y agitar enérgicamente durante unos minutos. </li><li> Lugar Shrinky-dink molde en una pequeña placa de Petri. Vierta la mezcla de PDMS hasta llegar a aproximadamente 1 / 2 cm sobre la superficie del molde. </li><li> Introducir la cápsula en la campana de vacío para eliminar todas las burbujas de la mezcla de PDMS. </li><li> Introducir la cápsula en el horno a 70 ° C, durante la noche. </li><li> Cortar sólidos PDMS del molde y se unen a un portaobjetos de vidrio con sólo aplicar presión. </li><li> Primer descarte de micropocillos-chip, ya que cuenta con restos de tinta incrustada entre PDMS. </li><li> Repita este procedimiento para producir un segundo chip que está sin tinta y tiene una forma más definida. </li><li> Limpie micropocillos-chip con solución al 70% de etanol. Colócalo debajo de la fuente de luz UV durante 10 minutos para esterilizarlo. </li></ol> 3. Las células de trampeo en micropocillos <ol><li> Recuento de células y se diluye en medio de cultivo a la concentración deseada (dependiendo del número de células iniciales en los pozos que le gustaría). Por ejemplo, para obtener aproximadamente 10-15 células por pocillo (media = 11, SD = 5,4, la tasa de carga = 93%), se utilizó una concentración de 8 × 10 4 células / ml. Para una concentración de 17 × 10 4 células / ml, se podría obtener de forma fiable entre 25 y 35 células por pocillo (media = 27.17857 SD = 7,7, la tasa de carga = 100%). </li><li> Con cuidado, coloque chips pocillos en un tubo de centrífuga de 50 ml que contiene una base de PDMS solidificado. </li><li> Añadir alrededor de 2JDP ml de la solución de células. </li><li> Centrifugar durante 5 minutos a 760 rpm y 4 ° C. </li><li> Pipeta de exceso de solución y lave cuidadosamente pocillos con PBS solución 1X. </li><li> Colocar en una placa de micropocillos Petry pequeño, con cuidado, teniendo el chip del tubo de centrífuga. </li><li> Lavar el exceso de células usando una solución de PBS X. </li><li> Lugar de chips de micropocillos bajo un microscopio invertido para verificar el número previsto de células por pocillo. </li><li> Incubar micropocillos que contienen las células en condiciones normales. <img src="/files/ftp_upload/old/692_2008_7_21_1/692_Figure-01.png" alt="Figura-01" width="350" height="183" /></li></ol> 4. Incubación de células <ol><li> Seguir el protocolo normal de EB en el laboratorio. </li><li> Cambiar el medio lentamente desde el lado de la cámara, no molestar a la celda de la microplaca. </li></ol>

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, 862-869 (1995).</li><li>Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+development+of+blastocyst-derived+embryonic+stem+cell+lines:+Formation+of+visceral+yolk+sac,+blood+islands+and+myocardium.">The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.</a> Journal of Embryology and Experimental Morphology. 87, 27-45 (1985).</li><li>Park, J., Cho, C. H., Parashurama, N., Li, Y., Berthiaume, F., Toner, M., Tilles, A. W., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation.">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7, 1018-1028 (2007).</li><li>Koike, M., Sakaki, S., Amano, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+embryoid+bodies+of+mouse+embryonic+stem+cells+formed+under+various+culture+conditions+and+estimation+of+differentiation+status+of+such+bodies.">Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.</a> J Biosci Bioeng. 104, 294-299 (2007).</li><li>Hwang, N. S., Varghese, S., Elisseeff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+differentiation+of+stem+cells.">Controlled differentiation of stem cells.</a> Advanced Drug Delivery Reviews. 60, 199-204 (2008).</li><li>Adelman, C. A., Chattopadhyay, S., Bieker, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP/BMPR/Smad+pathway+directs+expression+of+the+erythroid-specific+EKLF+and+GATA1+transcription+factors+during+embryoid+body+differentiation+in+serum-free+media.">The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.</a> Development. 129, 539-549 (2002).</li><li>Tanaka, N., Takeuchi, T., Neri, O. V., Sills, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum/cell+free+culture+system:+applications+and+preliminary+results+in+a+murine+model.">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> J Transl Med. 4, (2006).</li></ol>

Hemos desarrollado un método de cultivo sencillo, rápido y escalable para la carga de pre-definidos del número de células en los pozos microfabricated (moldeados a partir de Shrinky-Dinks) y mantenimiento para el desarrollo del cuerpo embrioides. Por último, estas células son fácilmente accesibles para su posterior análisis y la experimentación. Este método se presta a cualquier laboratorio y no requiere equipo especializado, ya que elimina la necesidad de fotolitografía. Podemos variar el tamaño de las cúpulas, así como la co...

Para los medios de comunicación MESC (McCloskey laboratorio protocolo): (por los medios de comunicación total preparado: 50ml, 100ml) KO DMEM: 40.8ml; 81.6ml KSR 15%: 7,5 ml, 15 ml 1x Penn-estreptococo: 0.5 ml, 1 ml 2 mM L-glutamina: 0,5 ml, 1 ml AANE: 0.5 ml, 1 ml LIF: 100 ul; 200ul BMP-4 (10ng/ml): 50 ul, 100 ul diluido BME:. 50 ul, 100 ul (Agregar 35ul de BME filtrado estéril de 5 ml de PBS y filtro de jeringa de esterilizar Descartar después de 2 semanas. La concentración final en la solución es 0,1 mM)

shrinky-dink hanging drops: a simple way to form and culture embryoid bodies

Cuerpos embrioides (EB) son agregados de células madre embrionarias. La forma más común de la creación de estos agregados es el método de la gota colgante, un enfoque laborioso de pipeteo un número arbitrario de las células en placas. Las interacciones entre las células madre obligado a cerca de uno al otro promueve la generación de la EBS. Debido a que los medios de comunicación en cada uno de los pozos tiene que ser intercambiados de forma manual todos los días, este enfoque es manual intensivo. Además, debido a los parámetros ambientales como la célula-célula, las células interacciones de los factores solubles, pH, y la disponibilidad de oxígeno pueden ser funciones del tamaño de EB, las poblaciones de células obtenidas a partir de gotas colgantes tradicionales pueden variar considerablemente, incluso cuando se cultivan en condiciones idénticas. Estudios recientes han demostrado claramente que el número inicial de células que forman el conjunto puede tener efectos significativos sobre la diferenciación de células madre. Hemos desarrollado un método de cultivo sencillo, rápido y escalable para la carga de pre-definidos del número de células en los pozos microfabricated y mantenimiento para el desarrollo del cuerpo embrioides. Por último, estas células son fácilmente accesibles para su posterior análisis y la experimentación. Este método se presta a cualquier laboratorio y no requiere equipo especializado. Se demuestra que este método mediante la creación de cuerpos embrioides con una línea roja fluorescente de células de ratón (129S6B6-F1).

Watch this Scientific Journal Video about Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies at JoVE.com

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Nos muestran un método sencillo y rápido para cargar previamente definidos el número de células en los pozos microfabricated y mantenimiento para el desarrollo del cuerpo embrioides.

Shrinky-Dink colgante Gotas: Una manera sencilla de Forma y Cultura cuerpos embrioides

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

shrinky-dink-hanging-drops-simple-way-to-form-culture-embryoid

Research

JoVE Journal

Biology

14.9K vistas.  University of California Merced - UC Merced. Nos muestran un método sencillo y rápido para cargar previamente definidos el número de células en los pozos microfabricated y mantenimiento para el desarrollo del cuerpo embrioides.

Video: Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Investigación

Biología

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

El análisis de las células madre pluripotentes utilizando criosecciones de cuerpos embrioides

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Técnica sencilla y eficaz para la preparación de suspensiones de células testiculares

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

El método de cultivo de la rebanada por Siguiendo desarrollo de gérmenes de dientes en Explante Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Método simple de ADN de fluorescencia<em&gt; In Situ</em&gt; Hibridación a los cromosomas aplastado

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

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

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

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

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

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

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Un análisis Simple reportero basado en fluorescencia para identificar los componentes celulares necesarios para la toxina de ricina una cadena (RTA) trata de levadura

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Un método Simple para el aislamiento de protoplastos de soja y aplicación al análisis de expresión génica transitoria

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.