Os autores gostariam de agradecer Glen Slocum (Medical College of Wisconsin) e Colleen A. Lavin (Nikon Instruments, Inc.) para a assistência técnica excelente, com experimentos de microscopia. Gregory Blass é reconhecida pela revisão crítica do manuscrito. Esta pesquisa foi apoiada pelos Institutos Nacionais de Saúde HL108880 concessão e American Diabetes Association conceder 1-15-BS-172 (para AS), eo Ben J. Lipps Research Fellowship da Sociedade Americana de Nefrologia (para DVI).

O uso de animais e bem-estar devem aderir ao Manual do NIH para o Cuidado e Uso de Animais de Laboratório seguindo protocolos revistos e aprovados pelo Comitê de Cuidado e Uso do animal Institucional (IACUC). 1. Lave rim <ol><li> Use 8 a 12 semanas de idade de ratos macho (sugerido é uma estirpe Sprague Dawley, no entanto outras estirpes de idade e sexo diferente pode ser utilizado com modificações apropriadas). </li><li> Anestesiar o animal de acordo com o procedimento permitido pelo protocolo IACUC; monitorar a profundidade da anestesia e inspecionar o animal. A descrição detalhada da cirurgia a ser realizada em 1,3-1,8 pode ser encontrado em Ilatovskaya et al 7. </li><li> Após anestesia adequada, colocar o animal em uma mesa cirúrgica, com temperatura controlada, fazer uma incisão na linha média do abdome (até 3 polegadas de comprimento), e descobrir a veia cava ea aorta. </li><li> Insira ligadura ao redor dos celíacos e artérias mesentérica superior e da abdominalaorta acima daqueles; não ligadura. </li><li> Blunt dissecar a aorta abdominal abaixo das artérias renais, e coloque duas ligaduras em torno dele, mas não ligar, em seguida, apertar a aorta acima das ligaduras e amarre o fio inferior. </li><li> Cateterize aorta com uma tubagem de polietileno PE50 (ligado a uma bomba seringa cheia com PBS) abaixo da braçadeira e fixar o cateter com a segunda ligadura; remover o grampo, ligar a bomba, e ligar a aorta e artérias mesentérica com doença celíaca. Rapidamente fazer incisão na veia renal para aliviar a pressão. </li><li> Infundir a aorta com o PBS pré-arrefecida durante 2 ou 3 minutos a um caudal de 6 ml / min. </li><li> Pare de perfusão, especiais de consumo e 7 decapsulate os rins, e colocá-los no gelo na solução PBS. Eutanásia do animal de acordo com o protocolo aprovado pelo IACUC. </li></ol> 2. Isolamento do Rato Os glomérulos <ol><li> Preparar 30 ml de solução fresca de 5% de BSA em meio RPMI 1640. </li><li> Usando uma lâmina de barbear e tesouras,isolar o córtex de ambos os rins, e em seguida, mediu até ficar homogénea. Este procedimento foi descrito anteriormente 7. </li><li> Empurrar o tecido moído durante o passo anterior através de um peneiro de aço inoxidável de malha 100 (pré-embebidos em solução de BSA a 5% / RPMI) utilizando uma espátula. Recolhe-se o fluxo de passagem e por força da gravidade permitir o fluxo de passagem para passar através de um peneiro de 140 mesh. </li><li> Filtra-se o fluxo através recolhidos a partir de 140 utilizando um peneiro de malha pré-embebido peneira de malha 200, descarta-se o filtrado e lava-se a parte superior do crivo com 10 - 15 ml da solução de BSA / RPMI preparado para recolher os glomérulos que sedimentam sobre o crivo. </li><li> Coloque a solução de BSA / RPMI contendo glomérulos sobre gelo em um tubo de 15 ml e deixar o sedimento glomérulos na parte inferior do tubo de até 20 min. O concentrado glomérulos no fundo do tubo irá ser claramente visto. Remover o excesso de solução, deixando cerca de 2 ml no tubo. </li></ol> 3. Single-channel patch-clamp Electrophysiology <ol><li> Prepare 5 x 5 milímetros chips de vidro capa de revestimento com 70.000 MW - 150.000 poli-ʟ-lisina, e deixe secar. Use cerca de 30 ml de solução estéril filtrado 0,01% em água por tampa de vidro. </li><li> Aquecer as soluções experimentais para a TA e encher a câmara de patch-clamp e pipeta. Para a monitorização canais trpC, usar uma solução de banho, em mM: 126 de NaCl, 1 CaCl2, 10 de HEPES, 2 de MgCl2, 10 de glucose, pH 7,4; pipeta: 126 de NaCl, CaCl2 1,5, HEPES 10, glucose 10; pH 7,4. <ol><li> Adicionar inibidores para a solução da pipeta para bloquear a actividade de canais endógenos, os quais não são relevantes para os estudos (recomendados são: 100 uM de ácido niflúmico ou DIDS (para bloquear Ca 2+ -activated canais Cl -), 10 mM de TEA (para inibir a grande condutância Ca 2 + - K + dependente do canal), 10 nM iberiotoxin (para bloquear os canais de K + -activated Ca2 +), 10 mM nicardipina (para bloquear N-tipo Ca2 + canais)) diretamente antes do experimento patch-clamp. </li></ol></li><li> Misture suavemente a solução contendo glomérulos, e em seguida, aplicar cerca de 50 l de la à tampa revestida chips de vidro poli-ʟ lisina. Deixe os glomérulos anexar por aproximadamente 5 min. </li><li> Mover os chips de vidro com glomérulos para a câmara de patch-clamp pré-cheia com a solução de banho; perfundir a câmara a um caudal de 3 ml / min durante 1 min para assegurar a remoção dos glomérulos não acopladas. </li><li> Realizar um experimento patch-clamp convencional em um modo ligado à célula 7. Com uma pipeta de vidro (7-10 mohms resistência pipeta) formar uma vedação de alta resistência entre uma pipeta e uma membrana podócitos por aplicação de sucção suave (uma pipeta ligada a um podócitos na superfície do glomérulo isolado é mostrado na Figura 2, em a esquerda). </li><li> Para as medições ligado de células, de baixo passam as correntes em 300 Hz por um filtro de oito pólos Bessel. </li><li> VOCÊse isolado glomérulos em experimentos de patch-clamp para até 4-6 horas. Mantenha a fração estoque glomérulos no gelo. </li></ol> 4. Raciometrico confocal de fluorescência As medições da concentração de cálcio intracelular no Podócitos <ol><li> Colocar 500 mL da fracção de glomérulos (descrito em 2.5) em 0,5 ml de tubo cónico e adicionar corantes cálcio Fura Red, AM e Fluo-4, AM. Utilizar 2 mM e 1 mM concentrações de banco de Fura Red, AM e Fluo-4, AM, respectivamente (armazenar a -20 ° C, dissolveu-se em DMSO) e 2,5 ul de usar cada corante por 500 ul de fracção de glomérulos. Imediatamente após a adição dos corantes cobrir o tubo com folha de alumínio. </li><li> Colocar os tubos num agitador rotativo durante pelo menos 20 minutos até 1 hora à TA. Nota: Os agentes farmacológicos podem ser adicionados durante este passo. </li><li> Prepare lamelas de vidro, cubra-os com poli-ʟ-lisina e permitir a secagem usando set placa aquecida a 70 ° C. </li><li> Uma vez que o carregamento de corantes de cálcio é complete, aplicar 100 ul de solução contendo os glomérulos para as lamelas revestidas com poli-ʟ-lisina e deixá-los aderir à superfície durante cerca de 5 min. Montar as lamelas anexado-glomérulos em uma câmara de imagem, e perfundir com a solução de banho (contendo (em mM): 145 NaCl, 4,5 de KCl, 2 de MgCl2, 10 HEPES, pH 7,35) com um caudal de 3 ml / min para remover os glomérulos acopladas e os corantes restantes. </li><li> Definir o microscópio confocal de varrimento a laser a um comprimento de onda de excitação de 488 nm e filtros de emissão (525/25 e 650/25 nm para Fluo-4 e Fura Red, respectivamente). Definir o software de imagem para uma freqüência e resolução desejada. </li><li> Encontre o glomérulos em claro e, em seguida, ligue a detecção do sinal de fluorescência. Ajustar a intensidade do laser para cada corante para evitar a saturação do sinal. Escolha o plano focal com as podocytes que estão diretamente ligados ao vidro. Isto minimiza o efeito causado pela contracção de um glomérulo em resposta à drogaaplicação. Verifique novamente se o glomérulo de escolha é bem ligado ao vidro; </li><li> Comece imaginando o plano focal de escolha (levar 512 x 512 imagens com a freqüência ajustada em 4 segundos para visualizar rápido Ca 2+ alterações transitórias. Use um / NA 1.4 ou lente objetiva semelhante 60X imagem de alta resolução para), aplicar medicamentos de interesse, e registar a resposta. <ol><li> Escolher um plano focal desejado (o mais próximo da superfície do vidro quanto possível, para assegurar a imagiologia de podócitos na superfície do glomérulo). Verifique a intensidade da fluorescência na Fluo4 e canais FuraRed, e certifique-se que o glomérulo é claramente visto em campo claro. </li><li> Iniciar a imagiologia. Antes da aplicação de todas as drogas, ficha, pelo menos, 1 min de linha de base de fluorescência para assegurar que o sinal é estável (não há picos repentinos ou desvanecimento do sinal). </li><li> Aplicar drogas desejadas com a ajuda de uma micropipeta; ter cuidado e garantir a droga foi capaz de difundir bem e alcançar o glomerulus. Misturar a solução de banho suavemente se necessário, monitorar o plano focal selecionado e verifique se ele não se moveu fora de foco por causa da aplicação de drogas. </li><li> Registar as alterações na intensidade de fluorescência para os sinais Fluo4 e FuraRed. Certifique-se a gravação é longo o suficiente, esperando até que o sinal atinge o nível de patamar ou atinge um pico e, em seguida, retorna à linha de base. Execute mudança solução ou adição de quaisquer outros medicamentos, se necessário. </li><li> Parar a gravação e salvar o arquivo no formato nativo do software. </li></ol></li></ol> 5. Análise de Imagem para as medições de cálcio <ol><li> Realizar análise de imagem com o software open ImageJ equipado com o plug-in utilitário ND que permite importar imagens no formato nativo ND2. </li><li> Importar a seqüência de imagens; certifique-se de dividir os canais e usar um modo hyperstack em tons de cinza. </li><li> Siga o → Ferramentas → caminho Analisar gerente ROI no software ImageJ para opena uma janela do gerenciador de ROI. Seleccione várias regiões de interesse (podocytes) usando uma ferramenta de seleção oval eo &quot;(t) Adicionar&quot; função no Gerenciador de ROI. Como o último ROI, selecione a área no fundo; salvar as ROIs selecionadas (Mais → Salvar). </li><li> Realce a janela que contém o canal selecionado para análises. Use a mais → função multi Medida no Gerenciador de ROI, marque a caixa &quot;uma linha por fatia&quot; no diálogo que aparece, e em seguida, clique em OK. Os resultados serão mostrados no formato que pode ser configurado na janela de resultados: entrar nas opções de menu Resultados → Definir Medidas, selecione &quot;valor de cinza média&quot; e calcular os valores de intensidade de pixel para cada ROI, que será exibido na janela de resultados em colunas separadas para cada ROI. </li><li> Copie os valores de intensidade de ROI medidos para cada canal (Fluo-4 e Fura Red) em software de análise de dados preferido; valores de intensidade fundo subtrair de cada datum ponto. </li><li> Para cada ponto de tempo calcular o rácio de intensidades do Fluo-4 aos canais Fura Red. Gráfico de dispersão / linha mudanças em tempo ponto de Ca 2+ transitória para cada ROI. Calcule os valores médios / SE para glomérulos selecionados. Nota: coluna Time deve ser definido de acordo com a frequência de imagem selecionada em 4.5. </li></ol> 6. Os cálculos de concentração intracelular de cálcio usando fluorescência de Fluo-4 Sinal <ol><li> Retirar uma amostra de glomérulos e executar o protocolo experimental acima para o passo 4.7. Depois de gravar a fluorescência de fundo adicionar Ionomicina (concentração final na câmara do banho deve ser de 10 uM) à solução de banho, e aumento da intensidade de fluorescência ficha. Uma vez que a intensidade atinge o seu máximo e começa a decadência, adicione MnCl2 (concentração final deve ser de 5 mm) para extinguir a fluorescência 8. </li><li> Analisar os dados obtidos em 6.1. Copie os Fluo-4 valores de intensidade de sinal ROI obtidos de acordocom o protocolo descrito em 5,1-5,5 por software de análise preferido. </li><li> Calcular a concentração intracelular de cálcio (em nM) na podócitos correspondente ao ROI através de uma fórmula: <img alt="Equação 1" src="/files/ftp_upload/52850/52850equation1.jpg" /> K onde d é uma dissociação pré-determinada constante para Fluo-4 (345 nM), F é a intensidade no ponto de tempo que está a calcular a concentração de cálcio para (linha de base), e F e F max min são os valores de intensidade na ponto de carga máxima de cálcio (após aplicação ionomicina) e após a extinção da fluorescência do (com MnCl 2), respectivamente (ver Figura 3). </li></ol>

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A abordagem aqui descrita permite a análise de manuseamento de cálcio pelos podócitos dos glomérulos de roedores. Esta técnica permite a aplicação de patch-clamp eletrofisiologia canal único e fluorescência confocal ratiometric imagem. No entanto, ambas as abordagens podem ser utilizadas separadamente, por conta própria. O protocolo proposto tem várias etapas relativamente simples, incluindo rubor 1) rim; 2) isolamento dos glomérulos por peneiração diferencial; 3) realização de patch-clamp experimentos e...

Rins manter o equilíbrio homeostático para várias substâncias e regular o volume de sangue de uma forma que determina a pressão arterial total. Distúrbios no filtração renal, reabsorção ou secreção de causar ou acompanham os estados patológicos, que variam de hiper ou hipotensão para a doença renal terminal que eventualmente exige transplante renal. A unidade de filtração renal (glomérulo) consiste em três camadas - o endotélio capilar, membrana basal e uma camada de célula única de células epiteliais - podócitos, que desempenham um papel importante na manutenção da integridade e função de um diafragma de fenda. Disfunção no filtro de permeabilidade selectiva glomerular provoca perda urinária de macromoléculas, tais como proteinúria. Vários agentes podem afectar a estrutura das podócitos e os seus processos de pé, que determinam a integridade da barreira de filtração glomérulos. Os podócitos estão envolvidos na manutenção do glomfunção de filtração eruli. Foi estabelecido que a manipulação imprópria de cálcio pelo podócitos leva a lesão celular e desempenha um papel importante na progressão de várias formas de nefropatias 2,3. Portanto, o desenvolvimento de um modelo que permite a medição direta da variação da concentração de cálcio intracelulares será fundamental para estudos de função podocyte. Isolado glomérulos foram anteriormente utilizados em um numerosos estudos, incluindo medição do coeficiente de reflexão albumina muda 4 e avaliação das correntes celulares integral na de células inteiras eletrofisiológicos medições de patch-clamp 5,6. No presente artigo descrevemos o protocolo que permite ao pesquisador para medir as mudanças de concentração de cálcio intracelular em resposta aos pedidos de agentes farmacológicos, estimar os níveis basais de cálcio dentro das células, e avaliar indivíduo atividade canais de cálcio. Medições da concentração de cálcio Ratometric e patch-clamp electrophysiology foram usadas para determinar as alterações na concentração intracelular de cálcio no seio da actividade podócitos e canal, respectivamente.

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			<td>Nikon confocal microscope (inverted)</td>
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			<td>Nikon</td>
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			<td>TMC</td>
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			<td>equipped with Faraday cage</td>
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			<td>Multichannel valve perfusion system</td>
			<td>AutoMake Scientific</td>
			<td>Valve Bank II</td>
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			<td>Recording/perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
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			<td>Molecular Devices</td>
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single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Aqui abordamos o problema de medir alterações agudas nos níveis de cálcio nos podocytes. A Figura 1 mostra uma representação esquemática do protocolo experimental projetado para realizar alta resolução de imagem confocal de fluorescência ao vivo e gravações de atividade de canais iônicos individuais nos podocytes do recém- glomérulos isolados de roedores. Resumidamente, após o rato é anestesiados, os rins devem ser lavados com PBS para limpá-las de sangue. Em seguida, os rins são exci...

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Single-channel Análise e cálcio Criação de imagem do Podócitos do recentemente isolados glomérulos

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Research

JoVE Journal

Biology

10.9K visualizações.  Medical College of Wisconsin. Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli. 

Vídeo: Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagem Funcional de gordura marrom em ratos com FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Biologia

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Avaliação do cálcio Faíscas intactas fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Quantificar Único microvasos Permeabilidade em perfusão isolada-Sangue Rat Lung Preparação

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Medindo o Coeficiente de Permeabilidade osmótica de água (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos planta isolada como um exemplo

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Implementando Grampo patch e ao vivo microscopia de fluorescência para monitorar propriedades funcionais de recentemente isolados PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

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 ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

A técnica de rim perfundidos rato isolado

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análise da função de células Beta utilizando imagens de cálcio de célula única resolução em ilhotas de Zebrafish

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medições simultâneas de cálcio intracelular e potencial de membrana no endotélio Cerebral rato recém isoladas e intacta

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...