O trabalho de Z Yang e Wang Y foi possível graças a conceder K02 AR051181 de NIAMS / NIH, subsídios da Muscular Dystrophy Association e Harvard Stem Cell Institute for Y Wang. O trabalho de Q Zeng e Xu X são suportados por uma bolsa concedida ao programa Lab imagem óptica do Brigham e do Hospital da Mulher. Os autores gostariam de agradecer Liu Wen do Departamento de Anestesia, BWH, para ajuda técnica no trabalho da célula.

Preparação celular <ol><li> Isolamento das células satélites <ol style="list-style-type:lower-alpha;"><li> Sob anestesia, os músculos dos membros de camundongos transgênicos GFP (C57BL / Ka-βactin-EGFP) são removidos. Após o corte em pedaços pequenos, as células satélite são enzimaticamente dissociado pela incubação do tecido picada com solução de 2% dispase colagenase durante 1 hora a 37 ° C. </li><li> Neutralize a digestão enzimática com meio de cultura contendo Ham F-10 e 20% FBS, dissociar o músculo por trituração repetido com uma pipeta Pasteur. As células são filtrados através de um filtro de células (ø70-100μm) e então semeadas em uma placa não-revestido por 1 hora a 37 ° C. </li><li> Remova cuidadosamente o sobrenadante e placa-los em um prato de colágeno revestido. Repita este passo uma hora mais tarde, para purificar os mioblastos. (Pré-plate) </li><li> Troque a mídia ea placa de células no tempo. Os mioblastos final purificado são cultivadas em meio contendo Ham F-10, 20% FBS, 100 U / ml de penicilina, estreptomicina e 100 mg / ml (média de crescimento), a 37 ° C com 5% de CO2 do ar, 95%. </li></ol> </li><li> Cultura e expandir a mioblastos primários em meio de crescimento com a adição de FGF básico de 10 ng / ml. </li><li> Para todos os experimentos in vivo, injetamos 5x10 5 células por músculo TA. </li><li> Ao colher os mioblastos, destacamos as células usando 0,25% de tripsina / EDTA e lavá-los com PBS duas vezes. O número de células são contadas com um hemocitômetro e as células são ressuspendidas em HBSS a uma concentração de 5x10 4 células / ml. As células concentradas em HBSS são colocados no gelo e injetado dentro de uma hora após a coleta. </li></ol> Vivo na imagem <ol><li> Camundongos machos mdx com a idade de 4-6 semanas são comprados a partir de Jackson laboratório e mantida em uma instalação de alojamento dos animais. </li><li> Após cerca de uma semana de alojamento para o meio ambiente, os ratos estão prontos para serem implantados com a cultura de células. </li><li> Antes do transplante das células, o mouse é anestesiado por uma injeção intraperitoneal (ip) da mistura de ketamina (100 mg / kg) e xilazina (10 mg / kg). Em seguida, o cabelo em torno da área do músculo TA é removida através da aplicação de Nair para a perna e à espera de 30 segundos para limpe-o com água destilada. </li><li> Enquanto o mouse está ainda sob anestesia, 5x10 5 células em HBSS 10μl estão lentamente injetadas em cada músculo TA em 3 pontos usando uma agulha de calibre 30. </li><li> Após o passo 4, o mouse pode ser regularmente fotografada pela fluorescência in vivo de imagens. A fluorescência de imagem da estação Nightowl LB981 (Berthold Technologies, Inc.), equipado com uma câmera de alta charge-coupled sensível, é usado para obter imagens das pernas dificultam dos ratos. </li><li> No dia da in vivo de imagens, nós primeiro verificar o crescimento do cabelo no músculo TA. Se necessário, voltar a aplicar Nair para remover o cabelo como descrito acima, depois de anestesiar o rato com a mistura de Ketamina e Xilazina. </li><li> Enquanto o mouse está sob anestesia, colocamos o mouse em uma posição de bruços em um pedaço de papel não-fluorescente preto (papel Artagain preto de Strathmore). Para expor totalmente o músculo TA para uma melhor imagem, que tape as patas traseiras para o papel. </li><li> Nós colocamos o mouse na câmara de imagem e posição da perna a ser trabalhada no centro do campo de visão da câmera. Nós primeiro mudar o filtro de emissão para tirar uma foto em tons de cinza fotográfica tradicional da perna com um tempo de exposição de 5 segundos. Reajuste a posição do mouse e foco da câmera, se necessário. Os parâmetros de imagem, incluindo o tempo de exposição (1.250 ms), altura de amostra (0,9 cm), etc são mantidas constantes no processo experimental. </li><li> Em seguida, interruptor no filtro de emissão adequada para o comprimento de onda da proteína verde fluorescente. Tomamos imagem de fluorescência com um tempo de exposição de 1.250 ms e um número binning de 1. Depois de imagem, o mouse é removido da câmara de imagem para uma gaiola que esperar a recuperação. </li><li> Para a análise, traçamos uma região de interesse (ROI) ao longo dos sinais fluorescentes para medir sua intensidade em termos de fótons por segundo. Também medimos o tamanho do ROI em termos de número de pixels como um outro valor de quantificação. Os experimentos de imagem pode ser repetida a cada semana ou mais freqüentemente ao longo de alguns meses, se necessário, para aquisição de dados longitudinal. </li></ol> No final dos experimentos, o mouse é sacrificados eo músculo TA é colhida para análise histológica.

<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>

Montamos uma plataforma de imagem confiável e estável para acompanhar a fluorescência rotulados células transplantadas no músculo esquelético anfitrião neste estudo. GFP-rotulados mioblastos de camundongo GFP transgênicos representa um tipo de células-tronco adultas que serão candidatos para terapia celular no futuro. A manipulação constante, incluindo o número de células, injeção de células, fundo claro, a posição de imagem e de parâmetros de máquina é importante para a obtenção de imagem de alta qualidade e especialmente para análise quantitativa. Fluorescência de imagens tem muitas aplicações na pesquisa biomédica, uma vez que é não-invasivo, fácil de usar, e tem uma alta taxa de transferência. Além de imagens de fluorescência pode fornecer uma medida quantitativa com base na contagem de fótons, porém, devido à dispersão, atenuação e absorção, a luz não pode penetrar uma grande distância no tecido, que é uma lacuna da técnica. Esforços têm sido feitos para uso repórter vermelho ou infravermelho próximo para aumentar a profundidade de penetração da luz. Outra vantagem de imagens de fluorescência é que é traduzível para clínicas específicas, se o repórter fluorescente é aprovado para uso humano. Comparação com ressonância magnética, tomografia computadorizada, PET e, fluorescência de imagem tem alta flexibilidade, uma vez que não requer hardware caro e agentes de imagem. Um exemplo é a fluorescência baseada endoscópio e micro-endoscópio que têm sido utilizados em clínicas. Através da combinação de imagens de fluorescência com outras modalidades, esperamos alcançar a capacidade de adquirir tanto as informações anatômicas e funcionais sobre os processos biológicos subjacentes.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NightOwl LB981</td>
<td>&nbsp;</td>
<td>Berthold Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

Distrofias musculares são um grupo de doenças musculares degenerativas caracterizada pela perda progressiva de células musculares contráteis. Atualmente, não há tratamento curativo disponível. Os recentes avanços na biologia das células estaminais têm gerado novas esperanças para o desenvolvimento de terapias celulares eficientes com base para tratar estas doenças. Transplante de vários tipos de células-tronco marcadas com proteínas fluorescentes em músculos de modelos animais distróficos tem sido utilizado amplamente no campo. A técnica não-invasiva com a capacidade de rastrear o destino das células transplantadas longitudinalmente pode continuar a nossa capacidade de avaliar o enxerto muscular por células transplantadas com mais precisão e eficiência. No presente estudo, demonstramos que fluorescence in vivo de imagens é um método sensível e confiável para rastreamento de GFP transplantado (Proteína Fluorescente Verde) marcado com células em músculos esqueléticos do rato. Apesar da preocupação de fundo, devido à utilização de uma luz externa necessária para a excitação da proteína fluorescente, descobrimos que usando o mouse nuas ou eliminar o cabelo com reagentes depilação muito deste problema é eliminado. Usando uma câmera CCD, o sinal fluorescente pode ser detectado no músculo tibial anterior (TA) após a injeção de 5 x 10 5 células de ambos os camundongos transgênicos ou GFP eGFP transduzidas cultura de mioblastos. Para os músculos mais superficiais, como o extensor longo dos dedos (EDL), a injeção de células menos produz um sinal detectável. Intensidade do sinal podem ser medidos e quantificados o número de fótons emitidos por segundo em uma região de interesse (ROI). Uma vez que as imagens obtidas mostram limites claros demarcar a área enxertada, o tamanho do ROI pode ser medido também. Se as pernas são posicionadas de forma consistente o tempo todo, as mudanças no número total de fótons por segundo por músculo e do tamanho do ROI refletir as mudanças no número de células enxertadas eo tamanho da área enxertada. Portanto, as alterações no músculo mesmo ao longo do tempo são quantificáveis. In vivo técnica de imagem fluorescente foi usado principalmente para controlar o crescimento de células tumorogenic, nosso estudo mostra que é uma ferramenta poderosa que nos permite acompanhar o destino de células-tronco transplantadas.

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

Nós descrevemos um<em&gt; In vivo</em&gt; Fluorescência imagem de protocolo para acompanhar a regeneração muscular, GFP-rotulados mioblastos após o transplante em músculos esqueléticos de ratos saudáveis ​​e distrófica. Este protocolo pode ser adaptado para estudo da regeneração muscular de transplante de outros tipos de células e em outras condições muscular também.

Rastreamento de Dinâmica de enxerto muscular em animais de pequeno In Vivo Fluorescent Imagem

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 visualizações.  Brigham and Woman's Hospital. Nós descrevemos um In vivo Fluorescência imagem de protocolo para acompanhar a regeneração muscular, GFP-rotulados mioblastos após o transplante em músculos esqueléticos de ratos saudáveis ​​e distrófica. Este protocolo pode ser adaptado para estudo da regeneração muscular de transplante de outros tipos de células e em outras condições muscular também.

Vídeo: 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.

Medição in vivo Micro-circulação no músculo esquelético por Intra-vital Microscopia

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

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.

Rotulagem e hESCs hMSCs com óxido de ferro nanopartículas para Não-invasiva em Rastreamento vivo com ressonância magnética

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).

Imagens de células vivas de F-actina Dynamics via Microscopia 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.

Avaliação ex vivo de Fatigabilidade Contratilidade e Alternans em músculos esqueléticos isolados

 
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 Clonal Rastreamento de Stem hematopoiéticas e células progenitoras Marcado por cinco proteínas fluorescentes usando confocal e microscopia multifotônica

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 Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

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.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

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.

No Vivo controle de timócitos na Câmara Anterior do olho a Laser, microscopia eletrônica de varredura em tempo 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.

Examinação da dinâmica nuclear do fermento mitotic e Meiotic da fissão pela microscopia viva-pilha da fluorescência

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.

Dinâmica de oligomerização dos receptores de superfície celular em células vivas pela microscopia de fluorescência de reflexão interna total combinada com análise de número e brilho

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