Il lavoro di Z e Y Wang Yang è stato reso possibile da concedere K02 AR051181 da NIAMS / NIH, borse di studio da Distrofia muscolare e della Harvard Stem Cell Institute per Y Wang. Il lavoro di Zeng Q e X Xu sono supportati da una sovvenzione del programma assegnato al Laboratorio di Imaging Ottico dal Brigham e l&#39;ospedale delle donne. Gli autori desiderano ringraziare Liu Wen dal Dipartimento di Anestesia, BWH, per un aiuto tecnico al lavoro delle cellule.

Preparazione delle cellule <ol><li> Cella di isolamento satellitare <ol style="list-style-type:lower-alpha;"><li> Sotto anestesia, i muscoli degli arti di topi transgenici GFP (C57BL / Ka-βactin-EGFP) vengono rimossi. Dopo aver tagliato in piccoli pezzi, le cellule satellite sono dissociato enzimaticamente tramite l&#39;incubazione del tessuto macinato con una soluzione di collagenasi dispasi al 2% per 1 ora a 37 ° C. </li><li> Neutralizzare la digestione enzimatica con i media di crescita contenente Ham F-10 e il 20% FBS, dissociare il muscolo da triturazione ripetuto con una pipetta Pasteur. Le cellule vengono filtrati attraverso un colino cella (ø70-100μm) e poi placcato in maniera non rivestito piatto per 1 ora a 37 ° C. </li><li> Rimuovere delicatamente il supernatante e la piastra su un piatto rivestito di collagene. Ripetere questa 1 ora passo successivo, per purificare il mioblasti. (Pre-piastra) </li><li> Cambiare il supporto e la piastra delle cellule nel tempo. I mioblasti finale purificato sono coltivate in terreno contenente Ham F-10, 20% FBS, 100 U / ml penicillina e 100 mg / ml di streptomicina (mezzo di coltura) a 37 ° C con 5% di CO2, l&#39;aria del 95%. </li></ol> </li><li> Cultura ed espandere la mioblasti primari nel terreno di coltura con aggiunta di FGF base di 10 ng / ml. </li><li> Per tutti gli esperimenti in vivo, abbiamo iniettare 5x10 5 cellule muscolari per TA. </li><li> Durante la raccolta del mioblasti, si staccano le cellule utilizzando 0,25% tripsina / EDTA e lavate con PBS due volte. I numeri di cellulare vengono contati con un emocitometro e le cellule sono risospese in HBSS ad una concentrazione di 5x10 4 cellule / ml. Le cellule concentrate in HBSS sono immessi sul ghiaccio e iniettata entro 1 ora dopo la raccolta. </li></ol> Imaging in vivo <ol><li> Maschio topi mdx all&#39;età di 4-6 settimane sono acquistati da Jackson laboratorio e mantenuto in un impianto di stabulazione degli animali. </li><li> Dopo circa una settimana di alloggio per l&#39;ambiente, i topi sono pronti per essere impiantati con le cellule in coltura. </li><li> Prima del trapianto delle cellule, il mouse è anestetizzato da una intraperitoneale (ip) iniezione della miscela di ketamina (100 mg / kg) e xilazina (10 mg / kg). Poi i capelli intorno alla zona del muscolo TA viene rimosso applicando Nair alla gamba e in attesa di 30 secondi per pulirlo con acqua distillata. </li><li> Mentre il mouse è ancora sotto anestesia, 5x10 5 celle in HBSS 10μl sono iniettata lentamente in ogni muscolo TA a 3 punti con un ago da 30 gauge. </li><li> Dopo il punto 4, il mouse può essere regolarmente ripreso dal vivo in fluorescenza imaging. La fluorescenza immagini stazione Nightowl LB981 (di Berthold Technologies, Inc.), dotato di alta sensibilità accoppiamento di carica della telecamera, viene utilizzato per ottenere immagini dalla ostacolare zampe dei topi. </li><li> Il giorno di imaging in vivo, per prima cosa controllare la ricrescita dei capelli sul muscolo TA. Se necessario, riapplicare Nair per rimuovere i capelli come sopra descritto, dopo anestetizzare il mouse con la miscela di ketamina e xilazina. </li><li> Mentre il mouse è sotto anestesia, posizioniamo il mouse in posizione prona su un pezzo di nero non fluorescente carta (carta nera Artagain da Strathmore). Per esporre completamente il muscolo TA per immagini migliori, abbiamo nastro le zampe posteriori per la carta. </li><li> Abbiamo posto il mouse nella camera di imaging e la posizione delle gambe posteriori per essere ripreso al centro del campo visivo della telecamera. Per prima cosa cambiare il filtro per le emissioni di prendere un tradizionale scala di grigi dell&#39;immagine fotografica della gamba con un tempo di esposizione di 5 secondi. Regolare la posizione del mouse e messa a fuoco della fotocamera, se necessario. I parametri di imaging compreso il tempo di esposizione (1.250 m), altezza del campione (0,9 cm) ecc, sono mantenute costanti nel processo sperimentale. </li><li> Siamo quindi passare nel filtro emissione appropriati per la lunghezza d&#39;onda proteina fluorescente verde. Prendiamo l&#39;immagine di fluorescenza con un tempo di esposizione di 1.250 ms e un certo numero di binning 1. Dopo l&#39;imaging, il mouse viene rimosso dalla camera di imaging a una gabbia di aspettare il recupero. </li><li> Per l&#39;analisi, disegniamo una regione di interesse (ROI) oltre i segnali fluorescenti per misurare la loro intensità in termini di fotoni al secondo. Si misura anche la dimensione del ROI in termini di numero di pixel come un altro valore di quantificazione. Gli esperimenti di imaging può essere ripetuta ogni settimana o più frequentemente nel corso di un paio di mesi, se necessario, per acquisire dati longitudinali. </li></ol> Alla fine degli esperimenti, il mouse è eutanasia e il muscolo TA viene raccolto per l&#39;analisi istologica.

<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>

Abbiamo creato una piattaforma di imaging affidabile e stabile per il monitoraggio delle cellule trapiantate fluorescenza etichettati nel muscolo scheletrico ospitare in questo studio. GFP-etichettata da GFP mioblasti di topo transgenico cavalletti di un tipo di cellule staminali adulte che saranno candidati per la terapia cellulare per il futuro. Una manipolazione costante tra cui il numero delle cellule, l&#39;iniezione di cellule, sfondo chiaro, la posizione delle immagini e dei parametri della macchina è importante per ottenere immagini di alta qualità e soprattutto per l&#39;analisi quantitativa. Fluorescenza imaging ha numerose applicazioni nel campo della ricerca biomedica in quanto non è invasiva, facile da usare, ed ha un throughput elevato. Inoltre l&#39;imaging di fluorescenza in grado di fornire una misura quantitativa basata sulla conta fotone, anche se a causa della attenuazione dispersione e l&#39;assorbimento, la luce non può penetrare una grande distanza nel tessuto, che è una lacuna della tecnica. Gli sforzi sono in corso per usare giornalista rosso o vicino infrarosso per aumentare la profondità di penetrazione della luce. Un altro vantaggio di imaging di fluorescenza è che è traducibile alle cliniche se il giornalista specifico fluorescente è omologato per uso umano. Rispetto al MRI, CT, PET e, fluorescenza imaging ha un&#39;elevata flessibilità in quanto non richiedono hardware costosi e gli agenti di imaging. Un esempio è fluorescenza a base di endoscopio e micro-endoscopio che sono stati utilizzati nelle cliniche. Grazie alla combinazione di imaging di fluorescenza con altre modalità, ci aspettiamo di raggiungere la capacità di acquisire informazioni sia anatomiche e funzionali sui processi biologici sottostanti.

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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

Distrofie muscolari sono un gruppo di malattie muscolari degenerative caratterizzata dalla progressiva perdita di cellule muscolari contrattili. Attualmente, non esiste alcuna cura disponibile. I recenti progressi nella biologia delle cellule staminali hanno generato nuove speranze per lo sviluppo di terapie cellulari basate efficace per il trattamento di queste malattie. Trapianto di vari tipi di cellule staminali etichettati con proteine ​​fluorescenti nei muscoli di modelli animali distrofici è stato utilizzato ampiamente nel settore. Una tecnica non invasiva con la capacità di seguire il destino delle cellule trapiantate longitudinalmente può ulteriormente la nostra capacità di valutare l&#39;attecchimento del trapianto da cellule muscolari trapiantate più accurato ed efficiente. Nel presente studio, abbiamo dimostrato che in vivo l&#39;imaging di fluorescenza è un metodo sensibile e affidabile per il monitoraggio GFP trapiantati (proteina fluorescente verde)-etichettati cellule nei muscoli scheletrici del mouse. Nonostante la preoccupazione di fondo per l&#39;utilizzo di una luce esterna necessaria per l&#39;eccitazione di proteine ​​fluorescenti, abbiamo scoperto che utilizzando il mouse nude o eliminando i capelli con reagenti depilazione molto di questo problema è eliminato. Utilizzando una fotocamera CCD, il segnale fluorescente può essere rilevato nel tibiale anteriore (TA) muscolare dopo l&#39;iniezione di 5 x 10 5 cellule sia da topi transgenici GFP o eGFP trasdotte cultura mioblasti. Per i muscoli più superficiali come l&#39;estensore lungo delle dita (EDL), l&#39;iniezione di un minor numero di cellule produce un segnale rilevabile. Intensità del segnale possono essere misurate e quantificate come il numero di fotoni emessi al secondo in una regione di interesse (ROI). Dal momento che le immagini acquisite mostrano chiari confini che delimitano la zona innestata, la dimensione del ROI può essere misurata come bene. Se le gambe sono posizionate in modo coerente ogni volta, i cambiamenti nel numero totale di fotoni al secondo per muscolare e la dimensione del ROI riflettere le variazioni del numero di cellule trapiantate e le dimensioni della zona innestata. Pertanto, il cambiamento nel muscolo stesso nel corso del tempo sono quantificabili. In vivo tecnica di imaging a fluorescenza è stata utilizzata principalmente per rintracciare la crescita delle cellule tumorogenic, il nostro studio dimostra che è un potente strumento che ci consente di tenere traccia il destino delle cellule staminali trapiantate.

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

Ci descrivono un<em&gt; In vivo</em&gt; Protocollo di imaging di fluorescenza di monitorare la rigenerazione muscolare GFP-etichettata dopo trapianto di mioblasti scheletrici nei muscoli di topi sani e distrofici. Questo protocollo può essere adattato a studio rigenerazione muscolare da trapianto di altri tipi di cellule e in altre condizioni muscolari così.

Monitoraggio Dinamica di attecchimento muscolare nei piccoli animali da In Vivo Fluorescente Imaging

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 Views.  Brigham and Woman's Hospital. Ci descrivono un In vivo Protocollo di imaging di fluorescenza di monitorare la rigenerazione muscolare GFP-etichettata dopo trapianto di mioblasti scheletrici nei muscoli di topi sani e distrofici. Questo protocollo può essere adattato a studio rigenerazione muscolare da trapianto di altri tipi di cellule e in altre condizioni muscolari così.

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.

In vivo micro-circolazione di misura nel muscolo scheletrico da Intra-vitale Microscopia

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

Ricerca

Biologia

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.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la 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).

Imaging cellulare dal vivo di F-actina Dynamics tramite microscopia a fluorescenza 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.

Vivo Valutazione Ex della contrattilità, affaticabilità e alternanza in isolati muscoli scheletrici

 
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 clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine ​​fluorescenti che utilizzano confocale e microscopia Multiphoton

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 inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali 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.

Inseguimento In Vivo dei timociti nell'alloggiamento anteriore dell'occhio di microscopia a scansione Laser in tempo reale

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.

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

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.

Dinamica di oligomerizzazione dei recettori delle superfici cellulari nelle cellule viventi mediante microscopia a fluorescenza totale di riflessione interna combinata con l'analisi del numero e della luminosità

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