Damos las gracias a Haruya Ohno, Shinoda Kosaku, Sharp Louis, Tomoda Emi, y Lauren Ruiz de discusión, ayuda técnica y asistencia editorial sobre el manuscrito. Este trabajo fue apoyado por becas de los NIH (DK087853), del Programa de Investigación Biomédica y de Breakthrough Asubio Pharm Inc. para SKULA fue apoyado por una COMPARTIR becas de doctorado de la Universidad de Copenhague y la UE FP7 proyecto Diabat (SALUD-F2 -2,011-278.373) a Lise Madsen y Kristiansen Karsten. También reconocemos la DERC centro subvención (NIH P30 DK063720).

1. Preparar Medio Digestión Hacer 5 ml por 5 ratones por tejido (aproximadamente 1 ml / 1 g de tejido adiposo). <ol><li> Pesar de las enzimas de digestión: - Colaginasa D: 1,5 u / ml (1108874103, 1 g, Roche, 70334223) - Dispase II: 2,4 u / ml (04942078001, 0.980 mg / lyo, Roche, 11466200) </li><li> Añadir 25 ml de PBS y mezclar bien para disolver </li><li> Añadir CaCl 2 justo antes de la digestión de los tejidos a una concentración final de 10 mM </li></ol> 2. Disecar el tejido adiposo de ratones <ol><li> La disección debe realizarse con cuidado, e inmediatamente después de los ratones (6-8 semanas de edad) se sacrificaron. El trabajo estéril con etanol rociado en la piel del ratón antes de abrirlo. Coloque el tejido directamente en PBS limpio, BAT y subcutánea interescapular separado inguinal WAT. </li><li> Para BAT interescapular: tiene el ratón colocado con la parte posterior enfrentado, corte a lo largo de la parte posterior abierta y todo el camino hasta el cuello. El marróntejido adiposo se encuentra justo debajo de la piel entre los hombros (interescapular). BAT se puede ver como dos lóbulos en forma de mariposa. Una fina capa de grasa blanca que cubre el BAT debe ser cuidadosamente eliminado. </li><li> Para subcutánea WAT ​​(inguinal WAT): quitar la piel. El inguinal se encuentra directamente debajo de la piel en ambos lados, comenzando casi en la parte posterior y va al lado, en el interior de los muslos y hacia abajo, hacia el testículo. </li></ol> 3. Cortar y digerir el tejido adiposo <ol><li> Rápidamente eliminar toda contaminación, como el pelo, el músculo esquelético y el tejido conectivo de las secciones de tejido. </li><li> Seque los tejidos rápidamente en un papel para no diluir medio disección con PBS y colóquelo en un plato seco. </li><li> Añadir medio de digestión (añadió CaCl 2 antes de la digestión) y picar el tejido en trozos pequeños. Mezclar bien pipeteando arriba y abajo con una pipeta de 5 ml. Transferir los tejidos picados en tubos de 50 ml con el resto de la digestión medio, mezclar aún más pipeteando arriba y abajo. </li><li> Colocación en 37 ° C con agitación constante a 150 rpm durante 40-50 min. Compruebe cada 10-15 minutos para asegurarse de que la digestión va bien y evitar el exceso de digestión. </li></ol> Nota: media digestión correcta y el tiempo son importantes. El tejido tiene que ser digerido bien, pero la digestión exceso puede dañar las células. 4. Suspensión de células filtro <ol><li> Detener la digestión por la adición de 5 ml de medio completo (DMEM/F12 que contiene FPS 10% y P / S). Las células deben ser casi completamente homogénea. Mezclar bien pipeteando. </li><li> Centrifugar a 700 xg durante 10 min. </li><li> SVF ahora puede ser visto como un sedimento de color marrón en el fondo del tubo. Aspirar la capa aceitosa en la parte superior de adipocitos maduros y la mayor parte de la capa de líquido - mantener el sedimento y se disuelven en 10 ml de medio completo. Mezclar bien. </li><li> Coloque un filtro de células (50-70 m de diámetro) en un nuevo 50<html> ml tubo y se filtra la suspensión celular. </li></ol> 5. Células de la placa <ol><li> Transferir la suspensión celular a un tubo de 15 ml y centrifugar a 700 xg durante 10 min. </li><li> Aspirar el medio y el sedimento vuelva a suspender en 10 ml de medio completo. Pipeta y mezclar bien. </li><li> Estimar cuántos platos se necesitan. Esto puede variar y depende del tamaño de los gránulos. Una regla general es de 2 placas de 10 cm a partir de 5 ratones para WAT ​​inguinal y una placa de 10 cm para las MTD. </li><li> Células de la placa en los platos recubiertos de colágeno (I + D) en medio completo </li><li> 1-2 h después de la siembra de las células, el medio aspirado, lavar dos veces con PBS y se añade medio fresco. Este paso es importante ya que puede eliminar las células rojas de la sangre, células inmunes y otros contaminantes. </li></ol> 6. Diferenciar las células <ol><li> Hacer mantenimiento y medios de inducción. </li></ol> Medio de mantenimiento Completar con medio ha añadido: ent &quot;&gt; La insulina, conc. 5 mg / ml (5 mg / ml, *** 100 l de ácido acético en 10 ml de H 2 O para preparar pH se acidifica el agua 2.5. disolver la insulina en el agua acidificada. stock en la tienda -20 ° C) 3,3 &#39;,5-triyodo-L-tironina (T3), conc. 1 nM (10 mM acciones, *** disolver T3 en 1N NaOH y se añade medio de acciones para hacer 10 m. Sigma cat # T-2877) Almacenar a 4 ° C, bien por una semana Medio de inducción Mantenimiento de medio que contiene los siguientes compuestos: Indometacina, conc final. 125 mM de stock (0,125 M en etanol, Sigma cat # I-7378). La indometacina se debe calentar a 60 ° C para disolver. Dexametasona, conc final. 2 mg / ml (2 mg / ml en etanol calcetín, Sigma cat # D-1756) 3-isobutil-1-metilxantina (IBMX), conc. 0,5 mM (0,25 M de stock en DMSO, Sigma cat # I-5879) La rosiglitazona, final conc. 0,5 mM(10 mM de existencias en DMSO, Sigma cat # R-2408) Nota: Asegúrese de inducción medio fresco cada vez. <ol start="2"><li> Grow adipocitos primarios a 95-97% de confluencia en medio completo (confluente pero no demasiado lleno) </li><li> Cambio de medio completo regular con medio de inducción (día 0) </li><li> Después de 48 horas (día 2), cambiar medio a medio de mantenimiento con rosiglitazona (0,5 mM) </li><li> Después de más de 48 horas (día 4), cambie al medio de mantenimiento fresco con rosiglitazona (1 mM) para 2-3 días adicionales. 6-7 días después de añadir el medio de inducción, las células están totalmente diferenciadas a células adiposas maduras y están llenos de gotas de aceite. Las gotas aparecerá alrededor de 3-4 días después de la inducción. </li></ol> Nota: Cambio medio cada 2-3 días hasta que las baterías están completamente diferenciados. Las células pueden ahora ser cosechado y la expresión de ARNm de UCP1 y otheR Brown adipocito específicos de los genes se puede medir por qRT-PCR. Western blot se utiliza para detectar proteínas.

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Los autores declaran que no tienen intereses en conflicto financieros.

Aquí presentamos un sistema fiable celular para estudiar el desarrollo de las células de color beige / brite en la enseñanza primaria adipocitos cultivados en ratones. En comparación con varias líneas celulares inmortalizadas disponibles, este sistema es probable que ofrezca mayor relevancia para el pardeamiento de la grasa blanca in vivo. A pesar de que el estudio de estos adipocitos primarios ofrece algunas ventajas, también existen algunas limitaciones y preocupaciones que ...

<html> La obesidad es el aumento dramáticamente en todo el mundo y ahora se considera uno de los problemas más graves para la salud pública 1. Esta condición está relacionada con un desequilibrio en la ingesta de energía relativa a los gastos y resultados en el exceso de energía almacenada en forma de lípidos en el tejido adiposo blanco (WAT). WAT ampliada se asocia con una mayor masa corporal y el peso, mientras que el tejido adiposo marrón tiene la capacidad de disipar el exceso de energía para producir calor. Por lo tanto BAT puede funcionar como protección contra el frío y la obesidad 2,3. Esto se consigue mediante desacoplamiento del transporte de electrones en la mitocondria por la disociación de proteínas 1 (UCP1). Esta proteína se considera una característica de termogénesis sin BAT en 3. Varios estudios realizados en años recientes han revelado que los humanos adultos tienen funcional 4-8 BAT y, en consecuencia, la manipulación de las MTD en los seres humanos puede ser una intervención terapéutica potencial en la lucha contra la obesidad y sus enfermedades relacionadas. jove_content &quot;pruebas&gt; actual indica que hay dos tipos de adipocitos marrones existen en los roedores;&quot; clásica &quot;o&quot; pre-existentes &quot;grasa parda se desarrolla durante la etapa prenatal y forma dedicados depósitos adiposos marrones en región interescapular y otros tejidos periféricos En la otra mano. , un &quot;inducible&quot; forma de grasa marrón (así llamadas células Brite o beige) se desarrolla durante la etapa post-natal y aparece intercaladas en el tejido adiposo blanco. Los dos tipos de adipocitos marrones también son separadas por diferentes orígenes del desarrollo. Si bien la pre-existente adipocitos marrones surgen de myoblastsic-Myf5 como precursores, los inducibles brite / beige células intercaladas en WAT surgen de un linaje no Myf5 9,10. Además, las vías de regulación de este tipo de célula es probable que sea diferente de la marrón Myf5 derivado adipocitos 11. El desarrollo de las células de color beige (es decir, &quot;cambio de color&quot; blanco de grasa) puede ser activada en respuesta a la exposición al frío y crónica aβ3-adrenérgicos agonistas o agonistas de PPAR en adultos 12-14. Las células de color beige / brite es probable que sean un objetivo terapéutico prometedor para la manipulación de balance global de energía y, potencialmente, puede convertirse en parte del tratamiento de la obesidad, por lo que es importante para comprender los mecanismos moleculares precisos y vías de señalización por señales ambientales que controlan el desarrollo de color beige las células. Para comprender el control molecular de la pardeamiento de la grasa blanca, en experimentos in vitro son los más adecuados como la diferenciación de los preadipocitos tiene lugar más bien de forma asíncrona y es difícil de detectar las células in situ 15. Aunque los estudios sobre el desarrollo de los adipocitos hasta ahora se han realizado principalmente en líneas celulares tales como, 3T3-L1 3T3-F442A o HIB1, estas líneas celulares parecen carecer de la firma molecular de las células de color beige. Por otro lado, los adipocitos primarios aislados desde la inyección subcutánea WAT ​​tienen más probabilidades de recapitular los process de pardeamiento de la grasa blanca de forma autónoma celular. Aquí se proporciona un protocolo para el aislamiento eficaz de la fracción estromal-vascular de tejidos adiposos y para inducir el bronceado de grasa blanca en respuesta a los agonistas de PPAR. Rosiglitazona se ha demostrado que es un mediador especialmente eficaz de pardeamiento en estas células. Como se sugirió anteriormente 16, este sistema celular se puede utilizar para servir a un sistema fiable celular para estudiar el desarrollo de las células de color beige / brite.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reactivo </td></tr><tr><td> Colaginasa D </td><td> Roche </td><td> 11088874103 </td><td></td></tr><tr><td> Dispasa II </td><td> Roche </td><td> 04942078001 </td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> Medio DMEM </td><td> Pescador </td><td> 10017-CV </td><td> Con 2,5 g / l de glucosa y L-glutamina sin piruvato de sodio </td></tr><tr><td> Insulina </td><td></td><td></td><td></td></tr><tr><td> T3 (3,3 &#39;,5-triyodo-L-tironina) </td><td> Sigma </td><td> T-2877 </td><td></td></tr><tr><td> La indometacina </td><td> Sigma </td><td> I-7378 </td><td></td></tr><tr><td> Dexametasona </td><td> Sigma </td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td> Sigma </td><td> I-5879 </td><td></td></tr><tr><td> Rosiglitazona </td><td> Sigma </td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipo </td></tr><tr><td> Colágeno platos recubiertos </td><td> BD </td><td> 354450 </td><td> Placas de 10 cm </td></tr><tr><td> 70 micras filtro </td><td> BD Falcon </td><td> 352350 </td><td> Celular colador, 70 m nylon 1/ea </td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

Adipocitos marrones tienen la capacidad de desacoplar la cadena respiratoria en las mitocondrias y disipar la energía química en forma de calor. Desarrollo de UCP1-positivas adipocitos marrones en los tejidos adiposos blancos (llamados células beige o brite) se induce por una variedad de señales ambientales tales como la exposición al frío crónica o por agonistas PPAR, por lo tanto, este tipo de célula tiene potencial como una diana terapéutica para tratamiento de la obesidad. Aunque la mayoría de inmortalizadas líneas de adipocitos no puede recapitular el proceso de &quot;color&quot; de la grasa blanca en cultivo, adipocitos primarios aislados de la fracción del estroma vascular en el tejido subcutáneo adiposo blanco (WAT) proporcionar un sistema celular fiable para estudiar el control molecular de desarrollo de las células beige / brite . Aquí se describe un protocolo para el aislamiento eficaz de los preadipocitos primarios y para inducir la diferenciación de células de color beige / brite en cultivo. El efecto de dorado se puede evaluar mediante la expresión de la grasa marrón selectivo marcaERS tales como UCP1.

Browning de adipocitos primarios se puede acceder mediante la medición de la expresión de ARNm de UCP1 y otros genes de la grasa marrón-específicos o selectivos de QRT-PCR. Presentado en la Figura 1 son los datos de expresión génica en el inguinal-derivados adipocitos primarios. Las células fueron inducidas a diferenciarse en la presencia de dos diferentes dosis de rosiglitazona en 50 nm y 500 nm, respectivamente. Un subconjunto de células se trató con forskolina en 10 mM durante 4 hor...

Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Preadipocitos primarios blancas aisladas de tejidos adiposos blancos en ratones se pueden diferenciar en células beige / brite. Se presenta aquí un sistema fiable modelo celular para estudiar la regulación molecular de &quot;color&quot; de la grasa blanca.

Aislamiento y diferenciación de las células del estroma vascular a células Beige / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

isolation-differentiation-stromal-vascular-cells-to-beigebrite

Research

JoVE Journal

Biology

38.4K vistas.  University of California, San Francisco. Preadipocitos primarios blancas aisladas de tejidos adiposos blancos en ratones se pueden diferenciar en células beige / brite. Se presenta aquí un sistema fiable modelo celular para estudiar la regulación molecular de "color" de la grasa blanca.

Video: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Aislamiento y suero animal expansión libre de cordón umbilical humano derivado de células mesenquimales del estroma (MSC) y endoteliales formadoras de colonias Células Progenitoras (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Investigación

Biología

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Aislamiento de CD146<sup&gt; +</sup&gt; Las células estromales mesenquimales de pulmón Residente de los pulmones de ratas

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Aislamiento de murino coronaria células musculares lisas vasculares

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Aislamiento, caracterización y diferenciación de células cardiacas desde el corazón de ratón adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Aislamiento, proliferación y diferenciación de células madre derivadas de tejido adiposo de macaco rhesus

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...