Este trabalho foi financiado pelo National Institutes of Health Grants AR055686 e AR060836 e pós-doutorado para AD pelo francês Associação de Distrofia Muscular (AFM). A facilidade de imagem celular utilizada neste estudo é apoiado pelo National Institutes of Health Grants HD040677.

1. Repair Membrana imagens de células Usando Granel (grânulo de vidro) Wounding Este protocolo permite a marcação separadamente as células feridos e aqueles que não conseguem curar. A quantificação destas populações de células requer a utilização de três condições: 1. Teste (C1) - As células são deixadas a reparar na presença de Ca2 +, 2. Controlo 1 (nenhuma lesão C2) - As células são incubadas na presença de Ca 2 +, mas não lesionado, e 3. Controlo 2 (sem reparação C3) - células são deixadas para reparar na ausência de Ca 2 +. <ol><li> Cultivar células de&gt; 50% de confluência em três lamelas estéreis e lavar C1 e C2, duas vezes com a CIM a 37 ° C e C3 com PBS a 37 ° C e lamelas de transferência de silicone de O-rings. </li><li> Adicione 100 ml de solução de dextrano FITC pré-aquecido no CIM em C1 e C2 ou em PBS em C3. </li><li> Ferir membrana plasmática em C1 e C3 por suavemente ondulado 40 mg contas de vidro sobre a lamela à temperatura ambiente por lamelas de inclinação manualmente para trás ediante 6-8 vezes em um ângulo de 30 °. Nota: Para lesões leves, garantir as contas de vidro estão espalhados de maneira uniforme, minimizando, assim, lesões repetidas. Para melhorar a reprodutibilidade do ferimento entre as amostras, simultaneamente, realizar a lesão de contas de vidro de diferentes amostras a ser comparadas. </li><li> Evitar mais rolamento de esferas, transferir todos os lamelas para uma incubadora de 37 ° C em CO 2 ambiente e permitir a reparação de prosseguir por 5 min. </li><li> Sem permitir que as esferas rolam, remover as esferas de vidro e de FITC-dextrano por lavagem com CIM (C1 e C2) ou PBS (C3), a 37 ° C. </li><li> Local lamelas de volta no anel O e adicionar 100 ml de solução TRITC dextrano pré-aquecido lisina solucionáveis ​​no CIM em C1 e C2 ou em PBS sobre C3. </li><li> Incubar a 37 ° C durante 5 min em ambiente de CO 2. </li><li> Lavar as lamelas duas vezes com pré-aquecido CIM e fixar com PFA a 4% durante 10 min à temperatura ambiente. </li><li> Lavar duas vezes com PBS e incuba-se durante 2 minutos à temperatura ambiente em corante Hoechst. </li><li> Wash samples duas vezes com PBS, montar em um slide usando a mídia de montagem e células de imagem usando um microscópio de epifluorescência. </li><li> Use as células de C2 para determinar a intensidade de coloração de fundo vermelho e verde e usar esse valor para limite os canais vermelho e verde para todas as amostras (C1-C3). </li><li> Pontuação número total de células que são a) verde (lesionado e reparado) e b) vermelho ou vermelho e verde (ferido, mas não conseguiram reparar) em amostras C1 e C3. </li><li> Contagem&gt; 100 células verdes para cada condição e expressam a fração de células que não conseguiram reparar como um por cento de todas as células lesadas (verde e vermelho). </li></ol> 2. Imagens ao vivo da cinética de reparo da membrana celular após lesão Laser <ol><li> Lave as células pré-aquecido com CIM e, em seguida, colocar a lamela na CIM com corante FM. </li><li> Coloque a lamela em um suporte no topo fase incubadora mantida a 37 ° C. </li><li> Seleccionar uma região de 2 1-2 mm da membrana da célula, e para irradiar &lt;, 10 mseg com o laser pulsado. Atenuar a potência do laser, através do software de 40-50% da potência de pico. Poder Optimal permite lesão consistente, mas não-letal e isso deve ser determinado por tentativa e erro para cada linha de instrumento individual e celular sendo usado. </li><li> Para monitorar o reparo, a imagem a cada 10 segundos em epifluorescência e campo claro, começando antes do ferimento e continuar por 3-5 min após lesão. Nota: Para qualquer controle reparação, repita os passos de 2,1-2,4 com as células feridos em PBS contendo corante FM. </li><li> Para quantificar a cinética de reparação, medir FM celular corante fluorescente e representar graficamente a alteração na intensidade (ΔF/F0) durante o curso de imagiologia. Esta informação deve ser em média de mais de 10 células em cada condição e representados graficamente como a média ou valor de célula individual, conforme necessário. </li></ol> 3. Imagem Granel (grânulo de vidro) Lesão Induzida Lysosomal Exocitose As amostras incluem seguintes células cultivadas para&gt; 50%confluência: 1. Test (C1) - Células autorizados a reparar na presença de Ca 2 +, 2. Controlo 1 (C2; Nenhuma lesão) - Células não lesionados nem incubadas com o anticorpo primário, e 3. Controle 2 (C3; No reparação) - Células autorizados a reparar na ausência de Ca 2 +. <ol><li> Lavar lamelas C1 e C2, duas vezes com a CIM a 37 ° C e C3 com PBS a 37 ° C, e transferi-los em silicone O-rings. </li><li> Adicione 100 ml de pré-aquecido lisina solucionáveis ​​dextrano TRITC no CIM em C1 e C2 e na PBS em C3. </li><li> Ferir membrana plasmática em C1 e C3 tal como no passo 1.3. </li><li> Evitar mais rolamento de esferas, transferir todos os lamelas para uma incubadora de 37 ° C em CO 2 ambiente e permitir a reparação de prosseguir por 5 min. </li><li> Remover as esferas de vidro e TRITC dextrano por lavagem das lamelas em meio de crescimento frio, assegurando novamente sem laminação das pérolas de vidro sobre as células. </li><li> Lavar as lamelas duas vezes com meio de cultura fria e transferir para os anéis de vedação. </li><li> Para C1 e C3, Adicione 100 ml de rato anticorpo anti LAMP1 rato em meio de crescimento completas frias e adicione a, meios de crescimento completo frio para C2. </li><li> Para permitir a ligação do anticorpo lamelas incubar durante 30 min a 4 ° C. </li><li> Lave as lamelas três vezes com CIM frio e corrigir todos lamelas com PFA 4% por 10 min à temperatura ambiente e depois enxaguar 3x com CIM. </li><li> Incubar todas as lamelas em 100 ul de solução de bloqueio durante 15 minutos a RT </li><li> Incubar todas as lamelas em 100 mL de Alexa Fluor 488 anticorpo anti rato durante 15 min a 4 ° C. </li><li> Lavar duas vezes com PBS e incubar durante 2 minutos à temperatura ambiente em Hoechst. </li><li> Lave as células duas vezes com PBS, montar em um slide usando a mídia de instalação e de imagem usando um microscópio de epifluorescência. </li><li> Usando imagens de células C2, determinar a coloração de fundo não específica no vermelho (TRITC dextrano) e verde (Alexa Fluor 488 anticorpos) canais e usar esses valores de coloração de fundo para limiar os canais vermelho e verde para todas as amostras (C1-C3). &lt;/ Li&gt; <li> Use as&gt; 100 rotulado vermelhas células de C1 e C3 para medir a intensidade de coloração LAMP1 (verde) em células lesadas. Para uma experiência de sucesso da coloração LAMP1 em células a partir de C3 será significativamente menor do que nas células de C1. </li></ol> 4. Imagens ao vivo da Membrana Celular Lesão Provocado Tráfico subcelular <ol><li> Fluorescente rotular o compartimento de interesse através da transfecção de repórter adequado (por exemplo, CD63-GFP para os lisossomas 8) ou utilizando corantes fluorescentes 9. </li><li> Para lisossomos de etiquetagem com corante fluorescente, incubar as células cultivadas para 50% de confluência em meio de crescimento contendo FITC-dextrano </li><li> Permitir dextrano a ser sujeita a endocitose, durante 2 horas (ou mais, conforme necessário para a rotulagem endossomal suficiente com dextrano pela linha celular de interesse) na incubadora de CO 2. </li><li> Lavar as células com meio de crescimento pré-aquecido e incubar em que, durante 2 horas numa incubadora de CO2 para permitir que todosendocytosed dextrano a se acumular no lisossomo. </li><li> Antes de imagem, lavar a lamela na pré-aquecido CIM e montar em um suporte lamela no último estágio incubadora a 37 ° C. </li><li> Realizar widefield (para circulação em todo o celular) ou TIRF (movimento na superfície da célula e exocitose) de imagem - as células que não têm lotado FITC dextrano lisossomos rotulados são ideais para a imagem do movimento e exocitose de lisossomos individuais. </li><li> Alinhando os lasers TIRF para microscópio de imagem: Use 60X ou 100X com objetivo&gt; 1,45 NA. Configure o ângulo de feixe de laser incidente TIRF usando a abordagem do fabricante. </li><li> Ferir a membrana celular por irradiação de uma pequena região (1-2 mm 2) para &lt;100 mseg, com o laser pulsado a 40-50% de atenuação. </li><li> Para monitorar a resposta do lisossoma para a célula lesão, células de imagem em 4-6 frames / seg por pelo menos 2 minutos ou mais, dependendo da dinâmica de exocitose em células de interesse. </li></ol>

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Lesão da membrana celular in vivo ocorre devido a uma variedade de factores de stress fisiológico e várias abordagens experimentais foram desenvolvidos para imitar estes. Estes incluem ferindo membrana celular das células aderentes raspando-los fora do prato ou por passagem através de um furo de 9,10 seringa estreita. Depois de tais lesões das células curar em suspensão e não adere à matriz extracelular, como sucede normalmente em tecido. Ainda outros, como o uso de poros formando toxinas a...

Membrana celular mantém a integridade das células, fornecendo uma barreira entre a célula e para o ambiente extracelular. Um estímulo químico, elétrico ou mecânico que exceder o limite fisiológico normal, assim como a presença de patógenos invasores cada um pode resultar em prejuízo para a membrana da célula e desencadear uma resposta celular subseqüente para reparar esse dano. Para sobreviver essas lesões na membrana celular, as células possuem um mecanismo eficiente para o reparo. Este mecanismo é dependente de cálcio e envolve o tráfico intracelular de proteínas, tais como anexinas e MG53, entre outros, bem como os compartimentos intracelulares tais como endossomas, lisossomas, Golgi e vesículas derivadas mitocôndrias para a membrana celular ferido 1-7. No entanto, os dados da sequência de acontecimentos moleculares e subcelulares envolvidos na reparação da membrana celular danificada permanece pouco compreendido. Resposta de reparação da célula podem ser agrupadas em orelhaly e respostas tardias. Respostas precoces, que ocorrem dentro de segundos a minutos o tempo de escala, são tremendamente importantes para determinar a natureza das respostas tardias que levam à reparação celular de sucesso ou morte celular. Ensaios de ponto final com base na análise bioquímica e celular em massa ajudaram a estabelecer o envolvimento de processos moleculares e celulares de reparação. Mas, devido à heterogeneidade e rapidez de resposta de reparação celular, ensaios de ponto final não fornecem os detalhes cinéticos e espaciais da seqüência de eventos que levam a reparar. Abordagens que permitem lesão controlada da membrana celular e permitem monitorar o reparo da membrana celular e respostas subcelulares associados a alta resolução espacial e temporal são ideais para tais estudos. Aqui, estas abordagens têm sido apresentadas. Dois dos protocolos descrevem abordagens para acompanhar a cinética em tempo real de reparação da membrana celular e as respostas intracelulares associados com o processo de reparação em células vivas seguintes inj a laserUry. Como complemento a estes ensaios baseados imagens de células vivas, os ensaios de ponto final também foram descritos que fornecem uma medida de base populacional para o monitoramento reparação de células individuais e as respostas subcelulares associados. Para demonstrar a sua utilidade destas abordagens têm sido utilizadas para controlar o tráfico e exocitose de lisossomos em resposta à lesão da membrana celular.

<table border="0" cellpadding="0" cellspacing="0" width="814" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H1387</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP410</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>FITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1820</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1/10 000 in PBS</td>
		</tr>
		<tr>
			<td>FM dye</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td>1 &#181;g/&#181;l in CIM or PBS</td>
		</tr>
		<tr>
			<td>FITC dextran</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>2 mg/ml in growth medium</td>
		</tr>
		<tr>
			<td>LAMP1&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-19992</td>
			<td>1/300 in growth medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 chicken anti-rat IgG</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>1/500 in blocking solution</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-545-86</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petridish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone O ring</td>
			<td>Bellco</td>
			<td>1943-33315</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>Bellco</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>12001-566</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>VWR</td>
			<td>MP1400-500H</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Penicillin/Sreptomycin</td>
			<td>VWR</td>
			<td>12001-350</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>5% chicken serum in CIM is used for blocking solution during immunostaining</td>
		</tr>
		<tr>
			<td>PBS (Ca2+ and Mg2+ free)</td>
			<td>VWR</td>
			<td>12001-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Olympus America, PA</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF illuminator&#160;</td>
			<td>Olympus America, PA</td>
			<td>Cell^TIRF (IEC60825-1:2007)</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence illuminator</td>
			<td>Sutter Instruments, Novato CA</td>
			<td>Lambda DG-4 (DG-4 30)</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Photometrics, Tucson, AZ</td>
			<td>Evolve 512 EMCCD&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and anaysis software</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Slidebook 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsed laser</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Ablate (3iL13)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokaihit, Japan</td>
			<td>INUBG2ASFP-ZILCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cell membrane injury and subcellular processes involved in repair

A capacidade das células para curar feridas é um processo celular fundamental, mas os mecanismos celulares e moleculares envolvidas na cura feridas células estão mal compreendidos. Aqui são descritos os ensaios para monitorar a capacidade e cinética de cura das células em cultura após a lesão localizada. O primeiro protocolo descreve uma abordagem ponto final baseado para avaliar simultaneamente reparo da membrana celular capacidade de centenas de células. O segundo protocolo descreve uma abordagem de imagem em tempo real para monitorar a cinética de reparo da membrana celular em células individuais, após lesão localizada com um laser pulsado. Como as células de cura feridas envolve o tráfico de proteínas específicas e os compartimentos intracelulares para o local da lesão, o terceiro protocolo descreve o uso de uma abordagem baseada ponto final acima para avaliar um tal evento tráfico (lisossomal exocitose) em centenas de células lesionadas e simultaneamente o último protocolo descreve o uso de lesões de laser pulsado em conjunto com micros TIRFcópia para monitorar a dinâmica de compartimentos subcelulares individuais em células lesadas em alta resolução espacial e temporal. Embora os protocolos aqui descrever a utilização dessas abordagens para estudar a relação entre a reparação da membrana celular e exocitose lisossomal em células musculares em cultura, podem ser aplicadas como tal, para qualquer outra célula de cultura aderente e compartimento subcelular de escolha.

Protocolos aqui descritas para imagens de células único são monitorar a capacidade e cinética de reparo da membrana celular (protocolos 1 e 2) eo tráfico subcelular e fusão de lisossomos durante a reparação (Protocolos 3 e 4). Protocolo 1 mostra um ensaio de massa que permite marcar todas as células lesionadas e identificar as células danificadas que não conseguiram reparar. Os resultados na Figura 1 mostram que, enquanto as células não lesionados (Figur...

Watch this Scientific Journal Video about Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair at JoVE.com

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

O processo de cura de feridas envolve as células tráfico de proteínas específicas e os compartimentos intracelulares para o local da lesão da membrana celular. Este protocolo descreve os ensaios para monitorar esses processos.

Imagens de células Lesão de Membrana e Processos Subcelulares envolvidas no reparo

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

imaging-cell-membrane-injury-subcellular-processes-involved

Research

JoVE Journal

Biology

14.9K visualizações.  Children's National Medical Center.  O processo de cura de feridas envolve as células tráfico de proteínas específicas e os compartimentos intracelulares para o local da lesão da membrana celular. Este protocolo descreve os ensaios para monitorar esses processos. 

Vídeo: Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualização de MG53 mediada Repair Membrana Celular Usando In vivo E In vitro Sistemas

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Biologia

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Exteriorização Rat Mesentério: Um modelo para investigar a dinâmica celulares envolvidos na angiogênese

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Viva Imagem Ensaio para Avaliação das Funções de Ca<sup&gt; 2 +</sup&gt; E esfingomielinase na reparação de Pore-formando feridas Tóxicas

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Estado fundamental depleção Super-resolução Imaging em células de mamíferos

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Imagem celular ao vivo super-resolução de estruturas subcelulares

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imagem óptica baseada em fotodiodo para dinâmica de rede de gravação com resolução de neurônio único em invertebrados não transgênicos

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

Isolando e Imagens Ao Vivo, Regiões de Marcapasso Intacta da Pelve Renal do Rato por Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Isolamento de células musculares lisas cardíacas e vasculares de zebrafish adulto, juvenil, larval e embrionário para estudos eletrofisiológicos

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

Imagem da migração de neutrófilos na pele de camundongos para investigar a remodelação da membrana subcelular em condições fisiológicas

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...