Le travail de Yang Z et Y Wang a été rendu possible par la subvention du K02 AR051181 NIAMS / NIH, les subventions des Muscular Dystrophy Association et de Harvard Stem Cell Institute for Y Wang. Le travail de Q et X Xu Zeng sont soutenus par un programme de subvention attribué au laboratoire d&#39;imagerie optique du Brigham and Women &#39;s Hospital. Les auteurs tiennent à remercier M. Wen Liu du département d&#39;anesthésie, BWH, pour une aide technique dans le travail de la cellule.

Préparation de cellules <ol><li> L&#39;isolement cellulaire par satellite <ol style="list-style-type:lower-alpha;"><li> Sous anesthésie, des muscles des membres des souris transgéniques GFP (C57BL / Ka-βactin-EGFP) sont supprimés. Après avoir coupé en petits morceaux, les cellules satellites sont enzymatiquement dissocié en incubant le tissu hachée avec une solution dispase collagénase 2% pendant 1 heure à 37 ° C. </li><li> Neutraliser la digestion enzymatique avec les milieux de croissance contenant Ham F-10 et de 20% de FBS, dissocier les muscles par trituration répétée avec une pipette Pasteur. Les cellules sont filtrés à travers une passoire cellule (ø70-100 microns) et ensuite étalées sur un plat non revêtus pendant 1 heure à 37 ° C. </li><li> Retirer délicatement le surnageant et la plaque de les sur un plat recouvert de collagène. Répétez cette étape 1 heure plus tard, pour purifier les myoblastes. (Pré-plaque) </li><li> Changer les médias et les plaques des cellules dans le temps. Les myoblastes final purifié sont cultivées dans un milieu contenant Ham F-10, 20% de FBS, 100 U / ml de pénicilline et 100 pg / ml de streptomycine (milieu de croissance) à 37 ° C avec 5% de CO2, 95% d&#39;air. </li></ol> </li><li> Culture et élargir les myoblastes primaires en milieu de croissance avec l&#39;ajout de FGF basique de 10 ng / ml. </li><li> Pour toutes les expériences in vivo, nous injectons 5x10 5 cellules par muscle TA. </li><li> Lorsque la récolte des myoblastes, nous détacher les cellules en utilisant 0,25% de trypsine / EDTA et les laver avec du PBS deux fois. Le nombre de cellules sont comptées avec un hémocytomètre et les cellules sont remises en suspension dans HBSS à une concentration de 5x10 4 cellules / ul. Les cellules concentrées dans HBSS sont placés sur la glace et injecté 1 heure après la collecte. </li></ol> L&#39;imagerie in vivo <ol><li> Homme souris mdx à l&#39;âge de 4-6 semaines sont achetés auprès de Jackson laboratoire et maintenue à une facilité de logement des animaux. </li><li> Après environ une semaine d&#39;hébergement à l&#39;environnement, les souris sont prêts à être implantés avec des cellules cultivées. </li><li> Avant la transplantation de cellules, la souris est anesthésiée par une intrapéritonéale (ip) d&#39;injection du mélange de kétamine (100 mg / kg) et de xylazine (10 mg / kg). Puis les cheveux autour de la zone du muscle TA est éliminé par l&#39;application de Nair à la jambe et d&#39;attendre 30 secondes pour l&#39;essuyer avec de l&#39;eau distillée. </li><li> Alors que la souris est toujours sous anesthésie, 5x10 5 cellules dans HBSS 10 ul sont injectés lentement dans chaque muscle TA à 3 points à l&#39;aide d&#39;une aiguille de calibre 30. </li><li> Après l&#39;étape 4, la souris peut être régulièrement imagés par imagerie de fluorescence in vivo. L&#39;imagerie de fluorescence station de NightOwl LB981 (Berthold Technologies, Inc), équipé d&#39;une haute sensibilité à couplage de charge appareil, est utilisé pour obtenir des images à partir des jambes d&#39;entraver des souris. </li><li> Le jour de l&#39;imagerie in vivo, nous avons d&#39;abord vérifier la repousse des cheveux sur le muscle TA. Si nécessaire, nous ré-appliquer Nair pour enlever les poils comme décrit ci-dessus, après avoir anesthésier la souris avec le mélange de kétamine et de xylazine. </li><li> Alors que la souris est sous anesthésie, nous plaçons la souris dans une position couchée sur un morceau de noir non fluorescent papier (papier noir Artagain de Strathmore). Pour bien exposer le muscle TA pour une meilleure imagerie, nous bande les pattes arrières sur le papier. </li><li> On place la souris dans la chambre de l&#39;imagerie et la position de la patte postérieure à imager au centre du champ de vision de la caméra. Nous avons d&#39;abord passer par le filtre d&#39;émission de prendre un classique de gris image photographique de la jambe avec un temps d&#39;exposition de 5 secondes. Réajuster la position de la souris et l&#39;orientation de la caméra si nécessaire. Les paramètres d&#39;imagerie, y compris le temps d&#39;exposition (1250 ms), la hauteur des spécimens (0,9 cm), etc sont maintenus constants dans le processus expérimental. </li><li> Nous avons ensuite passer dans le filtre d&#39;émission approprié pour la longueur d&#39;onde la protéine fluorescente verte. Nous prenons l&#39;image de fluorescence avec un temps d&#39;exposition de 1.250 ms et un certain nombre de binning 1. Après l&#39;imagerie, la souris est retirée de la chambre d&#39;imagerie pour une cage d&#39;attendre la récupération. </li><li> Pour l&#39;analyse, nous tirons une région d&#39;intérêt (ROI) sur les signaux fluorescents pour mesurer leur intensité en termes de photons par seconde. Nous mesurons également la taille du ROI en termes de nombre de pixels comme une autre valeur de quantification. Les expériences d&#39;imagerie peut être répété chaque semaine ou plus souvent en quelques mois, si nécessaire, d&#39;acquérir des données longitudinales. </li></ol> A la fin des expériences, la souris est euthanasié et le muscle TA est récolté pour une analyse histologique.

<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>

Nous mettons en place une plateforme d&#39;imagerie fiable et stable pour le suivi de la fluorescence étiquetés cellules transplantées dans le muscle squelettique d&#39;hôte dans cette étude. GFP-étiquetée myoblastes de souris transgéniques GFP représente un type de cellules souches adultes qui seront candidats pour la thérapie cellulaire à l&#39;avenir. Une manipulation constante, y compris le nombre de cellules, l&#39;injection de cellules, sur fond clair, la position de l&#39;imagerie et le paramètre machine est important pour gagner la haute qualité d&#39;image et surtout pour l&#39;analyse quantitative. L&#39;imagerie par fluorescence a de nombreuses applications dans la recherche biomédicale comme il est non invasif, facile à utiliser, et a un débit élevé. En plus de l&#39;imagerie de fluorescence peut fournir une mesure quantitative basée sur les comptages de photons, mais à cause de la dispersion, l&#39;atténuation et l&#39;absorption, la lumière ne peut pas pénétrer sur une grande distance dans les tissus, ce qui est une lacune de la technique. Des efforts ont été entrepris pour l&#39;utilisation journaliste de rouge ou le proche infrarouge pour augmenter la profondeur de pénétration de la lumière. Un autre avantage de l&#39;imagerie de fluorescence, c&#39;est qu&#39;il est traduisible dans les cliniques si le journaliste fluorescente spécifique est approuvé pour usage humain. Par rapport à l&#39;IRM, CT, et la TEP, imagerie de fluorescence a une grande flexibilité car elle ne nécessite pas de matériel coûteux et des agents d&#39;imagerie. Un exemple est basées sur la fluorescence endoscope et les micro-endoscope qui ont été utilisés dans les cliniques. En combinant l&#39;imagerie de fluorescence avec d&#39;autres modalités, nous nous attendons à atteindre la capacité d&#39;acquérir à la fois des informations anatomiques et fonctionnelles sur les processus biologiques sous-jacents.

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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

Les dystrophies musculaires sont un groupe de maladies musculaires dégénératives caractérisées par une perte progressive des cellules musculaires contractiles. Actuellement, il n&#39;existe aucun traitement curatif. Les avancées récentes en biologie des cellules souches ont généré de nouveaux espoirs pour le développement de thérapies cellulaires efficaces basées pour traiter ces maladies. La transplantation de différents types de cellules souches étiquetées avec des protéines fluorescentes dans les muscles de modèles animaux dystrophiques a été largement utilisé dans le domaine. Une technique non invasive avec la capacité de suivre le destin des cellules transplantées longitudinalement peuvent encore notre capacité à évaluer la prise de greffe de muscle par les cellules transplantées plus précise et efficace. Dans la présente étude, nous démontrons que la fluorescence in vivo d&#39;imagerie est une méthode sensible et fiable pour le suivi de la GFP transplantés (Green Fluorescent Protein) cellules marquées dans les muscles squelettiques de souris. En dépit de la préoccupation de fond dû à l&#39;utilisation d&#39;une lumière extérieure nécessaire à l&#39;excitation de la protéine fluorescente, nous avons constaté que ce soit en utilisant la souris nude ou en éliminant les cheveux avec des réactifs d&#39;épilation beaucoup plus de ce problème est éliminé. En utilisant une caméra CCD, le signal fluorescent peut être détecté dans le jambier antérieur (TA) du muscle après l&#39;injection de 5 x 10 5 cellules soit de souris transgéniques GFP ou EGFP transduites la culture des myoblastes. Pour des muscles plus superficielles telles que l&#39;extenseur commun des orteils (EDL), l&#39;injection de moins de cellules produit un signal détectable. L&#39;intensité du signal peut être mesurée et quantifiée comme le nombre de photons émis par seconde dans une région d&#39;intérêt (ROI). Depuis les images acquises montrent des limites claires délimitant la zone implantée, la taille de la ROI peut être mesuré aussi bien. Si les jambes sont placés systématiquement à chaque fois, les changements dans le nombre total de photons par seconde et par les muscles et la taille de la ROI de refléter les changements dans le nombre de cellules greffées et la taille de la zone greffée. Par conséquent, les modifications dans le même muscle au cours du temps sont quantifiables. In vivo technique d&#39;imagerie fluorescente a été utilisée principalement pour suivre la croissance des cellules tumorigène, notre étude montre qu&#39;il est un outil puissant qui nous permet de suivre le destin des cellules souches transplantées.

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

Nous décrivons une<em&gt; In vivo</em&gt; Imagerie de fluorescence protocole à suivre la régénération du muscle par la GFP-étiquetée myoblastes après la transplantation dans les muscles squelettiques des souris à la fois sain et dystrophiques. Ce protocole peut être adapté à l&#39;étude la régénération musculaire par transplantation d&#39;autres types de cellules et dans d&#39;autres conditions musculaires ainsi.

Suivi dynamique de prise de greffe musculaire chez les petits animaux par des In Vivo Fluorescent Imaging

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

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Research

JoVE Journal

Biology

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

9.7K vues.  Brigham and Woman's Hospital. Nous décrivons une In vivo Imagerie de fluorescence protocole à suivre la régénération du muscle par la GFP-étiquetée myoblastes après la transplantation dans les muscles squelettiques des souris à la fois sain et dystrophiques. Ce protocole peut être adapté à l'étude la régénération musculaire par transplantation d'autres types de cellules et dans d'autres conditions musculaires ainsi.

Vidéo: 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.

In vivo la micro-circulation de mesure dans le muscle squelettique par voie intra-vitale microscopie

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

Recherche

Biologie

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.

Étiquetage CSEh et hMSCs d&#39;oxyde de fer nanoparticules pour les non-invasives dans le suivi in ​​vivo avec IRM

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).

Imagerie de cellules vivantes de la F-actine fluorescente Dynamics via chatoiement de microscopie (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.

Évaluation ex vivo de la contractilité, fatigabilité et isolées du Alternans muscles squelettiques

 
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.

In vivo suivi clonale de cellules souches hématopoïétiques et progénitrices Marqué par cinq protéines fluorescentes confocale et multiphotonique Microscopie

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.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

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.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

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.

Real Time In Vivo suivi des Thymocytes dans la chambre antérieure de le œil par microscopie à balayage Laser

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 dynamique nucléaire de levure de fission mitotique et méiotique par fluorescence Microscopie à cellules vivantes

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.

Dynamique d'oligomerisation des récepteurs de surface cellulaire dans les cellules vivantes par microscopie de fluorescence interne totale combinée à l'analyse du nombre et de la luminosité

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