Nos gustaría dar las gracias a Katharina Cosker Courchesne y Stephanie útil para los debates.

Preparación de los reactivos <ol><li> Revestimiento de colágeno: el colágeno capa p35 placas de cultivo de tejidos y colocar en un horno a 37 ° C durante 2 días antes de engrasar los divisores. La concentración final de colágeno debe estar en 0,71 mg / ml diluida en 0,001 N HCl. A continuación, añadir 1 ml de la mezcla por placa. </li><li> Cargadores de grasa: A fin de llenar el cargador de grasa, una jeringa de 60 ml debe ser llenado con grasa de vacío Corning. Use la jeringa para llenar el cargador de grasa, lo envuelve en papel de aluminio y luego autoclave durante 45 minutos. </li><li> Separadores de teflón: los separadores se pueden volver a utilizarse después de cada experimento, pero primero debe limpiarse de forma correcta. Retire el separador de la placa, limpie toda la grasa restante y el lugar en ácido sulfúrico durante 2 días. Después de la eliminación del ácido, enjuagar con agua 3 veces, hacer hervir durante 20 minutos, deje que se seque, coloque en un recipiente de vidrio p100 Petri y autoclave durante 20 minutos. </li><li> N2-metil: Pesar 1,5 g de metilcelulosa y colocarlo en un frasco de 500 ml. Añadir una barra de agitación y autoclave durante 20 minutos en seco (a partir de este momento todo el trabajo debe ser estéril). A continuación, añadir 500 ml de suero libre de los medios de comunicación (N 2), y revuelva en una cámara frigorífica hasta que se disuelva. Alícuota en 50 ml conicals y congelar a -20 ° C. Para las acciones de trabajo, alícuota de una de las 50 conicals mL en tubos de 1 ml y congelar a -20 ° C. </li><li> DRG los medios de comunicación: DMEM, 5% de suero de caballo inactivado por calor, y el 1% de penicilina estreptomicina. </li><li> 100ng/mL DRGN los medios de comunicación: La concentración de acciones de ambas factor de crecimiento nervioso (NGF) y el cerebro-factor neurotrófico derivado (BDNF) es 1mg/ml. Diluir cada una de las neurotrofinas 1:10.000 en los medios de comunicación DRG. Las culturas pueden ser cultivadas en NGF solo, lo que altera el complemento de las neuronas que sobreviven en los cultivos. Nota: Cuando sea necesario, la concentración de (arabinósido de citosina) es AraC 1um y se utiliza a una concentración final de 0.3uM. Esto inhibe el crecimiento de las células de Schwann y la glía otros. </li><li> 10ng/ml DRGN los medios de comunicación: Diluir el 100ng/mL DRGN los medios de comunicación (1:10) con los medios de comunicación DRG. <img src="/files/ftp_upload/old/951_2008_10_27_6/951_Figure-01.png" alt="Figura 1" width="392" height="294" /> Figura 1. Herramientas necesarias para la puesta en marcha </li></ol> Configuración de las cámaras de compartimentos (iniciar este proceso de 1-2 días antes de la disección) <ol><li> Hacer un rasguño en el medio de un colágeno recubiertas p35 plato con un movimiento hacia afuera. </li><li> 30ul lugar de N2-metil-celulosa en el medio de la nada. Set platos a un lado hasta divisor es engrasada. </li><li> Conecte un adaptador de calibre 23 stub luer al cargador de grasa. Sujete el separador de teflón con un par de pinzas hemostáticas 90 º de ángulo y déjelo con el divisor hacia arriba con un microscopio. Trazar la divisoria con la grasa de asegurarse de que cada vez que el adaptador se coloca en un nuevo punto de partida se inserta el adaptador en la grasa del paso anterior para que haya una línea continua de grasa (ver diagrama). Una vez que la grasa se aplica a la división entera, a su vez uno de los platos preparados p35 boca abajo y colocarlo de modo que el N2-metil-celulosa es el compartimiento medio. Presione sobre el fondo del plato con un par de pinzas. Asegúrese de presionar en el interior de la división en las cuatro esquinas (arriba a la izquierda, abajo a la derecha, parte superior derecha, inferior izquierda, se indica en el diagrama de &quot;X&quot;). <img src="/files/ftp_upload/old/951_2008_10_9_2/951_Figure-02.png" alt="Figura 2" width="171" height="183" /> Figura 2: Pasos para engrasar el divisor Nota: Es importante que se mantenga la firmeza necesaria para que la grasa hace que el sellado completo con el plato, pero si la presión se agrega demasiado, los axones no se cruzarán en los compartimientos laterales. Levante el hemostatos, dale la vuelta y despinzar el divisor. Por último, coloque el plato con el divisor firmemente bajo el microscopio se centra en la parte inferior del compartimento del medio. Con el cargador de la grasa, hacer una pequeña barrera (0,25 cm), de modo que una vez que las células se colocan en el compartimiento medio que no pueda escapar. </li><li> Después de haber creado varias culturas, los medios de comunicación en lugar DRG cada uno de los compartimentos laterales y colocar en una incubadora en la que las células se mantendrá. Permita que las culturas a sentarse por varias horas y luego compruebe si hay fugas. Si los medios de comunicación se ha filtrado en el compartimiento del medio, entonces la cultura no se puede utilizar. Nota: La primera vez que el aprendizaje de esta técnica, es importante crear más cultivos que se necesitan para un experimento, ya que varios se gotean. <img src="/files/ftp_upload/old/951_2008_10_9_3/951_Figure-03.png" alt="Figura 3" width="254" height="191" /> Figura 3: &quot;Good vs fugas&quot; cultura </li></ol> El mantenimiento de las neuronas DRG en las culturas en compartimentos <ol><li> Día 1: Reemplazar DRG los medios de comunicación en los compartimientos del lado de los medios de comunicación 100ng/mL DRGN + AraC. Realizar la disección y añadir las células en el compartimiento central (100.000 células). </li><li> Día 2: Agregar 10ng/ml los medios de comunicación + AraC en el exterior de la división de teflón hasta que los medios de comunicación fluye sobre la barrera de grasa y fluido intercambio con el compartimiento central. </li><li> Día 3: Vuelva a colocar los medios de comunicación en los compartimientos de un lado a 100ng/mL DRGN omitiendo el AraC y alrededor de las con 10ng/ml DRGN omitiendo el AraC. </li><li> Día 6: Vuelva a colocar los medios de comunicación en los compartimientos de un lado a 1ng/ml + AraC y rodearse de los medios de comunicación DRG + AraC. </li><li> Día 9: El uso de la experimentación. <img src="/files/ftp_upload/old/951_2008_10_9_5/951_Figure-04.png" alt="Figura 4" width="267" height="148" /> Figura 4: las imágenes IHC de los cuerpos celulares y axones distales Nota: Al cambiar los medios de comunicación, es importante para aspirar el líquido de la parte superior del compartimento de cada lado. Además, nunca cambiar los medios de comunicación desde el compartimento del medio en sí, sólo desde la rodean, y dejar que fluya sobre la barrera de grasa en el centro. </li></ol>

<ol>
	<li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Control+of+the+Local+Environment+of+Somas+and+Neurites.">Independent Control of the Local Environment of Somas and Neurites.</a> Methods in Enzymology. 58, 302-307 (1979).</li><li>Watson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+use+the+Erk5+pathway+to+mediate+a+retrograde+survival+response.">Neurotrophins use the Erk5 pathway to mediate a retrograde survival response.</a> Nature Neuroscience. 4, 981-988 (2001).</li><li>Heerssen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+motors+transport+activated+Trks+to+promote+survival+of+target-dependent+neurons.">Dynein motors transport activated Trks to promote survival of target-dependent neurons.</a> Nature Neuroscience. 7, 596-603 (2004).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2, 599-605 (2006).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 4, 2128-2136 (2006).</li><li>Ure, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+transport+and+steady-state+distribution+of+125I-nerve+growth+factor+in+rat+sympathetic+neurons+in+compartmented+cultures.">Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.</a> J Neuroscience. 4, 1282-1290 (1997).</li></ol>

En este video, nos han demostrado cómo preparar y mantener cámaras de compartimentos para su uso en el cultivo de neuronas DRG. Hecho correctamente, este sistema permite la separación del cuerpo celular del axón con el fin de estudiar los mecanismos por los cuales las neurotrofinas señal a través de los axones largos. Dado que no es el aislamiento de fluidos entre los compartimientos, permite la estimulación selectiva o el tratamiento de un compartimiento sin los otros compartimientos se vean afectados. Culturas compartimentados cám...

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		<table border="1">
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>collagen</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354249</td>
<td>&nbsp;</td>
<tr>
<td>N2-methylcellulose 400CPS</td>
<td>Reagent</td>
<td>Sel-Win Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon divider</td>
<td>Other</td>
<td>Tyler Research</td>
<td>CAMP10</td>
<td>many other types of dividers are available</td>
</tr>
<tr>
<td>Pin rake</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-PR</td>
<td>&nbsp;</td>
<tr>
<td>Grease loader</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-GLSS</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Fisher</td>
<td>MT10017CV</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-01</td>
<td>&nbsp;</td>
<tr>
<td>BDNF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-02</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Reagent</td>
<td>Fisher</td>
<td>14-635-5D</td>
<td>&nbsp;</td>
<tr>
<td>AraC</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C-1768</td>
<td>&nbsp;</td>
<tr>
<td>23 gauge luer stub adapter</td>
<td>Tool</td>
<td>Fisher</td>
<td>427565</td>
<td>&nbsp;</td>
<tr>
<td>90&deg; angle hemostats</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-7035</td>
<td>&nbsp;</td>		</tbody>
		</table>
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preparation and maintenance of dorsal root ganglia neurons in compartmented cultures

Las neuronas se extienden axones que están muy lejos del cuerpo celular para inervar los tejidos diana, donde objetivo de factores de crecimiento derivados son necesarios para la supervivencia neuronal y la función. Las neurotrofinas son especialmente necesarias para mantener la supervivencia y la diferenciación de las neuronas sensoriales que inervan pero la pregunta de cómo estas neurotrofinas derivadas de objetivos comunicar al cuerpo celular de las neuronas que inervan ha sido un área de investigación activa por más de 30 años. El modelo más comúnmente aceptada de cómo las señales de neurotrofinas llegar al cuerpo de la célula se propone que la señalización endosomas llevar a esta señal de forma retrógrada a lo largo del axón. A fin de estudiar el transporte retrógrado, un sistema de cultivo fue concebido originalmente por Robert Campenot, en el que los cuerpos celulares están aislados de sus axones. La técnica de preparación de estas cámaras de compartimentos para el cultivo de neuronas sensoriales recapitula la estimulación selectiva de terminales de las neuronas que se produce la liberación in vivo de los siguientes derivados de objetivos neurotrofinas. Retrógrada eventos de señalización que requieren transporte a larga distancia microtúbulos retrógrada dependientes tienen importantes implicaciones para el tratamiento de enfermedades neurodegenerativas.

Watch this Scientific Journal Video about Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures at JoVE.com

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

A continuación se describe la técnica de preparación y mantenimiento de cámaras de compartimentos de las neuronas sensoriales de cultivo de los ganglios de la raíz dorsal.

Preparación y mantenimiento de las neuronas dorsales ganglios de raíz en las culturas con compartimientos

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

preparation-maintenance-dorsal-root-ganglia-neurons-compartmented

Research

JoVE Journal

Biology

22.3K vistas.  Dana Farber Cancer Institute. A continuación se describe la técnica de preparación y mantenimiento de cámaras de compartimentos de las neuronas sensoriales de cultivo de los ganglios de la raíz dorsal.

Video: Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparación de cultivos neuronales a partir de embriones de Drosophila Midgastrula Etapa

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Investigación

Biología

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparación de los cultivos disociados ratón neuronas corticales

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparación de balas de cañón de genes y transfección biolística de neuronas en cultivo Cortar

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

Preparación de 2-dGuo cultivos tratados con órganos timo

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

Protocolo para las neuronas simpáticas de rata cultivo Ganglios cervical superior (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparación de Aplysia Sensorio-motor culturas de células neuronales

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral sistema inducible Raíz en<em&gt; Arabidopsis</em&gt; Y maíz

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

Divergencia de la microbiota radicular en diferentes hábitats basada en redes de correlación ponderadas

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Un ensayo de quimiotaxis mejorado para la identificación rápida de quimioatrayentes rizobacterianos en exudados radiculares

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...