El trabajo de Wang y Yang Z Y fue posible por la subvención K02 AR051181 de NIAMS / NIH, las donaciones de la Asociación de Distrofia Muscular y el Harvard Stem Cell Institute for Y Wang. El trabajo de Q y Zeng Xu X son apoyados por un programa de subvención concedido al Laboratorio de Óptica de la Imagen del Hospital Brigham y de Mujeres. Los autores desean agradecer a Liu Wen, del Departamento de Anestesia, BWH, en busca de ayuda técnica en el trabajo celular.

Preparación de células <ol><li> Aislamiento de células satélite <ol style="list-style-type:lower-alpha;"><li> Bajo anestesia, los músculos de los miembros de la GFP ratones transgénicos (C57BL / Ka-βactin-EGFP) se retiran. Después de cortar en trozos pequeños, las células satélite están disociadas enzimáticamente por incubación del tejido picado con una solución de colagenasa dispasa 2% durante 1 hora a 37 ° C. </li><li> Neutralizar la digestión enzimática con medios de cultivo que contienen Ham F-10 y el 20% de SFB, se disocian del músculo por trituración repetida con una pipeta Pasteur. Las células se filtran a través de un colador de células (Ø70-100μm) y luego colocan en un plato sin revestimiento durante 1 hora a 37 ° C. </li><li> Retire con cuidado el sobrenadante y la placa en un plato recubierto de colágeno. Repita este paso una hora más tarde para purificar los mioblastos. (Pre-placa) </li><li> Cambiar el soporte y la placa de las células en el tiempo. Los mioblastos final purificado se cultivan en un medio que contiene Ham F-10, el 20% de SFB, 100 U de penicilina / ml, y 100 ug / ml de estreptomicina (medio de crecimiento) a 37 º C con CO2 al 5%, 95% de aire. </li></ol> </li><li> La cultura y ampliar los mioblastos primarios en medio de cultivo con adición de FGF básico de 10 ng / ml. </li><li> Para todos los experimentos in vivo, se inyecta 5x10 5 células por músculo TA. </li><li> En la cosecha de los mioblastos, que separar las células mediante el uso de 0,25% de tripsina / EDTA y lavar con PBS dos veces. El número de células se contó con un hemocitómetro y las células se resuspendieron en HBSS a una concentración de 5x10 4 células / microlitro. Las células concentradas en HBSS se colocan sobre hielo y se inyecta dentro de una hora después de la recolección. </li></ol> Imágenes in vivo <ol><li> Mujer ratones mdx a la edad de 4-6 semanas se compran a Jackson de laboratorio y mantenidos en un centro de alojamiento de los animales. </li><li> Después de una semana de alojamiento en el medio ambiente, los ratones están listos para ser implantados con las células cultivadas. </li><li> Antes del trasplante de células, el ratón está anestesiado por un intraperitoneal (ip) de la mezcla de ketamina (100 mg / kg) y xilazina (10 mg / kg). A continuación, el pelo alrededor de la zona del músculo TA se elimina mediante la aplicación de Nair en la pierna y esperar 30 segundos para limpiarla con agua destilada. </li><li> Mientras que el ratón está todavía bajo la anestesia, 5x10 5 células en HBSS 10μl se inyecta lentamente en cada músculo TA a los 3 puntos con una aguja de calibre 30. </li><li> Después del paso 4, el ratón se puede regular mediante imágenes en vivo de imágenes de fluorescencia. La imagen de fluorescencia estación Nightowl LB981 (Tecnologías de Berthold, Inc.), equipado con una alta sensibilidad de acoplamiento de carga de la cámara, se utiliza para obtener imágenes de las piernas obstaculizar de los ratones. </li><li> En el día de imágenes in vivo, en primer lugar comprobar la regeneración del cabello en el músculo TA. Si es necesario, volver a aplicar Nair para quitar el pelo como se describe anteriormente, después de anestesiar el ratón con la mezcla de ketamina y xilazina. </li><li> Mientras que el ratón está bajo anestesia, se coloca el ratón en una posición boca abajo en un pedazo de negro no fluorescente de papel (papel negro Artagain de Strathmore). Para exponer el músculo por completo de asistencia técnica para una mejor imagen, grabamos las patas traseras en el papel. </li><li> Ponemos el ratón en la cámara de imagen y la posición de la pata trasera a ser reflejado en el centro del campo de visión de la cámara. En primer lugar, cambiar el filtro de emisiones para tomar la tradicional escala de grises imagen fotográfica de la pierna con un tiempo de exposición de 5 segundos. Reajuste la posición del ratón y el enfoque de la cámara si es necesario. Los parámetros de imagen, incluyendo el tiempo de exposición (1.250 ms), altura de la muestra (0.9 cm), etc se mantienen constantes en el proceso experimental. </li><li> A continuación, cambiar el filtro de emisiones correspondientes a la longitud de onda proteína verde fluorescente. Tomamos la imagen de fluorescencia con un tiempo de exposición de 1.250 ms y un número de intervalos de 1. Después de la filmación, el ratón se retira de la cámara de imágenes a una jaula para esperar la recuperación. </li><li> Para el análisis, se dibuja una región de interés (ROI) a través de las señales fluorescentes para medir su intensidad en términos de fotones por segundo. También medimos el tamaño del retorno de la inversión en términos de número de píxeles como otro valor de la cuantificación. Los experimentos de imagen se puede repetir cada semana o con mayor frecuencia durante unos meses, si es necesario, para obtener datos longitudinales. </li></ol> Al final de los experimentos, el ratón es la eutanasia y el músculo TA se cosecha para su análisis histológico.

<ol>
	<li>Ballou, B., Ernst, L. A., Waggoner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+tumors+in+vivo.">Fluorescence imaging of tumors in vivo.</a> Curr Med Chem. 12, 795-805 (2005).</li><li>Green, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+as+a+marker+for+gene+expression.">fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Godavarty, A., Sevick-Muraca, E. M., Eppstein, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fluorescence+lifetime+tomography.">Three-dimensional fluorescence lifetime tomography.</a> Med Phys. 32, 992-1000 (2005).</li><li>Graves, E. E., Weissleder, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+molecular+imaging+of+small+animal+tumor+models.">Fluorescence molecular imaging of small animal tumor models.</a> Curr Mol Med. 4, 419-430 (2004).</li><li>Graves, E. E., Yessayan, D., Turner, G., Weissleder, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+in+vivo+fluorochrome+concentrations+measured+using+fluorescence+molecular+tomography.">Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography.</a> J Biomed Opt. 10, 44019-44 (2005).</li><li>Gurfinkel, M., Ke, S., Wen, X., Li, C., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+optical+imaging+and+tomography.+Dis+Markers.">Near-infrared fluorescence optical imaging and tomography. Dis Markers.</a> , 107-121 .</li><li>Lee, J., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fluorescence+enhanced+optical+tomography+using+referenced+frequency-domain+photon+migration+measurements+at+emission+and+excitation+wavelengths.">Three-dimensional fluorescence enhanced optical tomography using referenced frequency-domain photon migration measurements at emission and excitation wavelengths.</a> J Opt Soc Am A Opt Image Sci Vis. 19, 759-771 (2002).</li><li>Xu, X., Yang, Z., Liu, Q., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fluorescence+imaging+of+muscle+regeneration+by+transplanted+eGFP-labeled+myoblasts.">In vivo fluorescence imaging of muscle regeneration by transplanted eGFP-labeled myoblasts.</a> , (1985).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology+of+cancer+cells+visualized+with+fluorescent+proteins.">In vivo cell biology of cancer cells visualized with fluorescent proteins.</a> Curr Top Dev Biol. 70, 121-144 (2005).</li><li>Coulton, G. R., Morgan, J. E., Partridge, T. A., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+skeletal+muscle+myopathy:+I.+A+histological,+morphometric+and+biochemical+investigation.">The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation.</a> Neuropathol Appl Neurobiol. 14, 53-70 (1988).</li><li>Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells.">Muscle satellite cells.</a> Int J Biochem Cell Biol. 35, 1151-116 (2003).</li><li>Mendell, J. R., Kissel, J. T., Amato, A. A., King, W., Signore, L., Prior, T. W., Sahenk, Z., Benson, S., McAndrew, P. E., Rice, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+transfer+in+the+treatment+of+Duchenne's+muscular+dystrophy.">Myoblast transfer in the treatment of Duchenne's muscular dystrophy.</a> N Engl J Med. 333, 832-838 (1995).</li><li>Bogdanovich, S., Perkins, K. J., Krag, T. O., Khurana, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutics+for+Duchenne+muscular+dystrophy:+current+approaches+and+future+directions.">Therapeutics for Duchenne muscular dystrophy: current approaches and future directions.</a> J Mol Med. 82, 102-115 (2004).</li><li>Morgan, J. E., Coulton, G. R., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdx+muscle+grafts+retain+the+mdx+phenotype+in+normal+hosts.">Mdx muscle grafts retain the mdx phenotype in normal hosts.</a> Muscle Nerve. 12, 401-409 (1989).</li><li>Sherwood, R. I., Christensen, J. L., Weissman, I. L., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+skeletal+muscle+contributions+from+circulating+cells,+bone+marrow+cells,+and+hematopoietic+stem+cells.">Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells.</a> Stem Cells. 22, 1292-1304 (2004).</li><li>Cerletti, M., Jurga, S., Witczak, C. A., Hirshman, M. F., Shadrach, J. L., Goodyear, L. J., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient,+functional+engraftment+of+skeletal+muscle+stem+cells+in+dystrophic+muscles.">Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.</a> Cell. 134, 37-47 (2008).</li></ol>

Hemos creado una plataforma de imagen estable y fiable para el seguimiento de la fluorescencia etiquetados células trasplantadas en el músculo esquelético de acogida en este estudio. GFP-etiquetados mioblastos de las buenas prácticas agrarias de ratones transgénicos representa un tipo de células madre adultas que serán candidatos para la terapia celular en el futuro. Una constante manipulación, incluyendo el número de células, la inyección de células, fondo claro, la posición de la imagen y los parámetros de la máquina es importante para obtener imágenes de alta calidad y, sobre todo para el análisis cuantitativo. Fluorescencia de imagen tiene muchas aplicaciones en la investigación biomédica, ya que no es invasiva, fácil de usar, y tiene un alto rendimiento. Además de imágenes de fluorescencia puede proporcionar una medición cuantitativa basada en el recuento de fotones, aunque debido a la dispersión, atenuación y absorción, la luz no puede penetrar a una gran distancia en el tejido, que es un defecto de la técnica. Se han realizado esfuerzos para utilizar el reportero de color rojo o del infrarrojo cercano para aumentar la profundidad de penetración de la luz. Otra de las ventajas de imágenes de fluorescencia es que se puede traducir a las clínicas si el reportero fluorescente específica está aprobado para uso humano. En comparación con el MRI, CT y PET, fluorescencia de imagen tiene una gran flexibilidad, ya que no requiere hardware caro y agentes de imagen. Un ejemplo es la fluorescencia basada endoscopio y endoscopio micro-que se han utilizado en las clínicas. Mediante la combinación de imágenes de fluorescencia con otras modalidades, esperamos alcanzar la capacidad de adquirir información tanto anatómica y funcional de los procesos biológicos subyacentes.

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<td>NightOwl LB981</td>
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tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

Las distrofias musculares son un grupo de enfermedades musculares degenerativas caracteriza por una pérdida progresiva de las células contráctiles del músculo. En la actualidad, no existe ningún tratamiento curativo disponible. Los avances recientes en biología de células madre han generado nuevas esperanzas para el desarrollo de terapias basadas en células eficaces para tratar estas enfermedades. El trasplante de varios tipos de células madre marcadas con proteínas fluorescentes en los músculos de los modelos animales distróficos se ha utilizado ampliamente en el campo. Una técnica no invasiva con la capacidad de rastrear el destino de las células trasplantadas pueden longitudinalmente aún más nuestra capacidad para evaluar el injerto muscular, las células trasplantadas con mayor precisión y eficiencia. En el presente estudio, hemos demostrado que in vivo de fluorescencia de imagen es un método sensible y fiable para el seguimiento de las buenas prácticas agrarias trasplantado (Green Fluorescent Protein) marcado con células en los músculos esqueléticos del ratón. A pesar de la preocupación de fondo debido a la utilización de una luz externa necesaria para la excitación de la proteína fluorescente, se encontró que mediante el uso de cualquiera de ratón desnudo o la eliminación del pelo con los reactivos de depilación gran parte de este problema se elimina. Utilizando una cámara CCD, la señal fluorescente se puede detectar en el tibial anterior (TA) muscular después de la inyección de 5 x 10 5 células de cualquiera de las buenas prácticas agrarias ratones transgénicos o eGFP transduced cultura de mioblastos. Para que los músculos más superficiales como el extensor largo de los dedos (EDL), la inyección de menor número de células se produce una señal detectable. Intensidad de la señal puede ser medido y cuantificado el número de fotones emitidos por segundo en una región de interés (ROI). Puesto que las imágenes obtenidas muestran límites claros delimitar la zona implantada, el tamaño de la ROI se puede medir así. Si las piernas se colocan constantemente en todo momento, los cambios en el número total de fotones por segundo por el músculo y el tamaño de la ROI reflejar los cambios en el número de células injertadas y el tamaño de la zona injertada. Por lo tanto, los cambios en el mismo músculo en el tiempo son cuantificables. En vivo técnica de imagen fluorescente se ha utilizado principalmente para realizar el seguimiento del crecimiento de las células tumorogénico, nuestro estudio demuestra que es una poderosa herramienta que nos permite realizar el seguimiento del destino de las células madre trasplantadas.

Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Se describe un<em&gt; In vivo</em&gt; Imagen de fluorescencia protocolo para controlar la regeneración del músculo por las buenas prácticas agrarias con etiqueta de mioblastos después del trasplante en los músculos esqueléticos de los ratones sanos y distrófica. Este protocolo se puede adaptar para estudiar la regeneración del músculo mediante el trasplante de otros tipos de células y en otras condiciones musculares también.

Seguimiento de Dinámica de injerto muscular en pequeños animales por En Vivo Fluorescente de imágenes

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

tracking-dynamics-muscle-engraftment-small-animals-vivo-fluorescent

Research

JoVE Journal

Biology

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

9.7K vistas.  Brigham and Woman's Hospital. Se describe un In vivo Imagen de fluorescencia protocolo para controlar la regeneración del músculo por las buenas prácticas agrarias con etiqueta de mioblastos después del trasplante en los músculos esqueléticos de los ratones sanos y distrófica. Este protocolo se puede adaptar para estudiar la regeneración del músculo mediante el trasplante de otros tipos de células y en otras condiciones musculares también.

Video: Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

En vivo la micro-circulación de medición en el músculo esquelético de Intra-vital Microscopía

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Investigación

Biología

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etiquetado y hESCs hMSCs con nanopartículas de óxido de hierro para no invasivo en el seguimiento in vivo mediante RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imágenes de células vivas de la F-actina Dynamics a través de Microscopía Fluorescente Speckle (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Evaluación ex vivo de la contractilidad, fatigabilidad y alternancia en aislados músculos esqueléticos

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

En Tracking Clonal vivo de madre hematopoyéticas y células progenitoras Marcado por cinco proteínas fluorescentes utilizando confocal y Microscopía Multifotónica

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Seguimiento en Vivo de los timocitos en el compartimiento Anterior del ojo mediante láser, microscopía en tiempo real

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examen de la dinámica nuclear de levadura de ficción mitotica y meiótica por microscopía de células vivas de fluorescencia

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Dinámica de oligomerización de receptores de superficie celular en células vivas mediante la microscopía de fluorescencia de reflexión interna total combinada con análisis de número y brillo

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...