Nuestro trabajo es apoyado por el NIH subvenciones NS048447 y KD AG017216to. Li Liu gustaría agradecer al Dr. Heikki Tanila (Universidad de Kuopio, Kuopio, Finlandia) para su supervisión y apoyo durante el desarrollo del protocolo original.

Tirar del tubo capilar de vidrio <ol><li> El tubo capilar de vidrio se compra a La Inc Sutter Instrument (vidrio borosilicato, B100-75-10). </li><li> Tire de los tubos capilares en un Sutter P-87 Flaming micropipeta extractor, con el índice de calor en 300 y el índice de presión de 330. </li><li> Recorte el extremo del tubo capilar de vidrio con unas tijeras, de modo que la punta cónica tiene un diámetro interior de 0,5 mm. </li></ol> Cisterna magna técnica de punción de líquido cefalorraquídeo Muestras de LCR se han tomado de la cisterna magna (Figura 1) utilizando un método que se publicó anteriormente 1. <img src="/files/ftp_upload/old/960_2008_10_13_2/960_Figure-01.png" alt="Figura 1" width="432" height="184" /> Figura 1 1. Los ratones son anestesiados por la ketamina (100mg/kg) y xilazina (10mg/kg), administrado por vía intraperitoneal. En el momento de la inducción de la anestesia, los ratones se mantienen en una incubadora a 37 ° C. 2. La piel del cuello se afeita, y el ratón se coloca boca abajo en el instrumento estereotáxico con el contacto directo de una almohadilla eléctrica. Una sonda de temperatura rectal se inserta en el recto para que el calor producido por la almohadilla se ajusta en respuesta a los cambios de la temperatura corporal. La cabeza está protegida con los adaptadores de la cabeza. El lugar de la cirugía se limpia con povidona yodada al 10%, seguido por 7 0% de etanol (repetir 3 veces), y una incisión sagital de la piel se hace inferior a la protuberancia occipital. 3. Bajo el microscopio de disección, el tejido subcutáneo y músculos (m. Biventer cervicis de m. recto dorsal mayor capitis) son separados por una disección roma con una pinza. Un par de microretractors se utiliza para mantener los músculos de distancia. 4. El ratón se establece de manera que la cabeza forma un ángulo de casi 135 o con el cuerpo. 5. Bajo el microscopio de disección, la duramadre de la cisterna magna se presenta como un triángulo inverso brillante y clara a través del cual el bulbo raquídeo y un vaso sanguíneo principal (arteria espinales dorsales), y el espacio del LCR son visibles (Figura 2). <img src="/files/ftp_upload/old/960_2008_10_13_3/960_Figure-02.png" alt="Figura 2" width="304" height="307" /> Figura 2 6. La duramadre es borrado en seco con un hisopo de algodón estéril. Penetrar en el tubo capilar en el Citerna magna a través de la duramadre, lateral a la arteria espinales dorsales (Figura 2). Después de un cambio notable en la resistencia a la inserción de un tubo capilar, el LCR fluye dentro del tubo capilar. 7. Retire con cuidado el tubo capilar, y conectarlo a una jeringa de 3 ml a través de un tubo de polietileno que tiene un diámetro interno de 1 mm. Inyectar el líquido cefalorraquídeo en un pre-marcadas tubo eppendorf de 0,5 ml y congelar inmediatamente el tubo en hielo seco y luego la transfiere en una -80 ° C congelador. 8. Después de tomar muestras de LCR, los músculos se vuelven a alinear, y la piel se sutura (4-0, Ethicon, Johnson &amp; Johnson). Aproximadamente 1 ml de NaCl al 0,9% que se inyecta por vía subcutánea para evitar de deshidratación. El ratón se mantiene en la incubadora para mantener la temperatura corporal hasta que se recupere, el peso del ratón se controla un día y una semana después de la cirugía. Resultados y Discusión Se describe un protocolo de alta fiabilidad para la toma de muestras de LCR en serie de los ratones sin contaminación detectable en plasma. 1. El procedimiento completo generalmente toma 10 minutos por ratón (incluyendo la anestesia). El volumen de líquido cefalorraquídeo obtenido depende de las cepas de ratones. En el PS / APP ratones Tg doble usamos 2, el volumen promedio es de aproximadamente 5 l (3 a 7 l), mientras que el P301L (JNPL3) 3 ratones dan un rendimiento de alrededor de 10 l-15 l. Para el muestreo de serie, un máximo de 8.7 l puede tomar de manera segura cada vez que en un intervalo de 2-3 meses. 2. Durante la cirugía, es importante la posición de la cabeza y el cuerpo del ratón correctamente en el marco estereotáxico para que la duramadre de la cisterna magna puede estar expuesto lo suficiente. Evita cuidadosamente los vasos sanguíneos cuando penetra en la duramadre con el tubo capilar para prevenir la contaminación de las proteínas plasmáticas, que pueden ser monitoreados en la muestra de LCR por inmunotransferencia de ApoB 4. 3. Reducir al mínimo el daño a los tejidos durante cada cirugía es importante, ya que conglutinación tejido puede aumentar la dificultad por el sangrado de predisposición para la toma de muestras siguientes. 4. La temperatura del cuerpo necesita para mantenerse en buen estado durante la cirugía debido a una hipotermia causada por la anestesia puede afectar significativamente los niveles de tau fosforilada en el cerebro muy rápidamente 5. 5. El Ab ELISA protocolo y los anticuerpos que se utilizaron fueron descritas en detalle en Refolo et al. 6. Dos l de líquido cefalorraquídeo de los ratones PS / APP dará resultados satisfactorios cuando se diluye 1:50-1:60. ELISA para la tau, 4-5 l de LCR P301L (JNPL3) los ratones será suficiente para lala detección del total de tau, tau o fósforo ylated en treonina 231 (Los kits son adquiridos de Bio-Fuente). 6. Esta técnica se puede aplicar a otros modelos de ratón de las enfermedades neurológicas, por lo que un pequeño volumen de LCR es satisfactorio para los ensayos que desee.

<ol>
	<li>Liu, 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=Longitudinal+observation+on+CSF+Abeta42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF Abeta42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol Dis. 17, 516 (2004).</li><li>Holcomb, 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=Accelerated+Alzheimer-type+phenotype+in+transgenic+mice+carrying+both+mutant+amyloid+precursor+protein+and+presenilin+1+transgenes.">Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.</a> Nat Med. 4, 97 (1998).</li><li>Lewis, J., 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=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25, 402 (2000).</li><li>DeMattos, R. B., 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=Plaque-associated+disruption+of+CSF+and+plasma+amyloid-beta+(Abeta)+equilibrium+in+a+mouse+model+of+Alzheimer's+disease.">Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.</a> J Neurochem. 81, 229 (2002).</li><li>Planel, E., 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=Anesthesia+leads+to+tau+hyperphosphorylation+through+inhibition+of+phosphatase+activity+by+hypothermia.">Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.</a> J Neurosci. 27, 3090 (2007).</li><li>Refolo, L. 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+cholesterol-lowering+drug+reduces+beta-amyloid+pathology+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease.</a> Neurobiol Dis. 8, 890 (2001).</li></ol>

Se describe un protocolo de alta fiabilidad para la toma de muestras de LCR en serie de los ratones sin contaminación detectable en plasma. 1. El procedimiento completo generalmente toma 10 minutos por ratón (incluyendo la anestesia). El volumen de líquido cefalorraquídeo obtenido depende de las cepas de ratones. En el PS / APP ratones Tg doble que used2, el volumen promedio es de aproximadamente 5 l (3 a 7 l), mientras que el P301L (JNPL3) 3 ratones dan un rendimiento de alrededor de 10 l-15 l. Para el muestreo de serie, un má...

a technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse 

La enfermedad de Alzheimer (EA) es una enfermedad neurodegenerativa progresiva que es caracterizada patológicamente por depósito extracelular de péptidos β-amiloide (Aß) y la acumulación intraneuronal de proteína tau hiperfosforilada. Debido a que el líquido cefalorraquídeo (LCR) está en contacto directo con el espacio extracelular del cerebro, que proporciona un reflejo de los cambios bioquímicos en el cerebro en respuesta a procesos patológicos. LCR de pacientes con EA muestra una disminución de los 42 aminoácidos forma de Aß (Aß42), y el aumento de tau total y tau hiperfosforilada, aunque los mecanismos responsables de estos cambios aún no se entienden completamente. Transgénicos (Tg) los modelos de ratón de Alzheimer proporcionan una excelente oportunidad para investigar cómo y por qué los niveles de Aß y tau en el LCR cambio a medida que progresa la enfermedad. Este sentido, demuestran una refinada técnica de punción cisterna magna para el muestreo de la enfermedad por el ratón. Esta técnica de muestreo muy suave permite que las muestras de LCR en serie que se obtenga de el mismo ratón a intervalos de 2-3 meses, que minimiza en gran medida el efecto de confusión de la variabilidad entre el ratón en los niveles de Aß o tau, por lo que es posible detectar alteraciones sutiles en el tiempo. En combinación con Aß y ELISA tau, esta técnica será útil para los estudios diseñados para investigar la relación entre los niveles de CSF Aß42 y tau, y su metabolismo en el cerebro en modelos de ratón de Alzheimer. Los estudios en ratones Tg podría proporcionar validación importante en cuanto a la posibilidad de Aß CSF o los niveles de tau para ser utilizados como marcadores biológicos de progresión de la vigilancia de las enfermedades, y para controlar el efecto de las intervenciones terapéuticas. Como los ratones pueden ser sacrificados y los cerebros pueden ser examinadas por los cambios bioquímicos o histológicos, los mecanismos subyacentes a los cambios del LCR puede ser mejor evaluado. Estos datos pueden ser de carácter informativo para la interpretación de los cambios humanos AD CSF.

Watch this Scientific Journal Video about A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse at JoVE.com

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgénicos (Tg) los modelos de ratón de Alzheimer proporcionan una excelente oportunidad para investigar cómo y por qué los niveles de Aß y tau en el cambio CSF ​​conforme progresa la enfermedad en pacientes humanos. Este sentido, demuestran una refinada técnica de punción cisterna magna para el muestreo de LCR de serie del ratón.

Una técnica para la recolección de serie del líquido cefalorraquídeo de la cisterna magna en el ratón

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

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Research

JoVE Journal

Biology

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

100.7K vistas.  Columbia University.  Transgénicos (Tg) los modelos de ratón de Alzheimer proporcionan una excelente oportunidad para investigar cómo y por qué los niveles de Aß y tau en el cambio CSF ​​conforme progresa la enfermedad en pacientes humanos. Este sentido, demuestran una refinada técnica de punción cisterna magna para el muestreo de LCR de serie del ratón. 

Video: A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Vampiric Aislamiento de líquido extracelular de<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Investigación

Biología

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimiento de serie de la madre de espermatogonias y células progenitoras (SSC) en Cultura para la derivación de largo plazo de ratones adultos Líneas SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Aislamiento de ARN de ratón Páncreas: Un tejido de ribonucleasa-rica

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

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 ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

La Asamblea y la Aplicación de &#39;Shear Anillos&#39;: A Novel endotelial Modelo de Orbital, unidireccional y periódica flujo de fluido y la tensión de cizallamiento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La Técnica del riñón perfundido aislado del ratón

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Colección de células únicas de células trofoblastas en embriones humanos en fase de implantación

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.