Este trabalho foi financiado pelo NIH concede R37 AI34867 e R01 GM064625 a NWA Agradecemos ao Dr. R. Tweten da Universidade de Oklahoma para a expressão plasmídeo SLO.

1. Transcricional silenciamento de ASM <ol><li> Semente de 1,5 x 10 5 células HeLa em 2 ml de DMEM meio de crescimento (DMEM de alto teor de glucose com 10% de soro fetal bovino (FBS), 2 mM de L-glutamina, 1% de Penn-Strep) em pratos de fundo 35 milímetros de vidro (MatTek) e incubar durante a noite a 37 ° C numa atmosfera de 5% de CO2. </li><li> No dia seguinte, aspirado de fora os meios de crescimento e substituí-la com 2 ml de DMEM reduzida meio de soro (DMEM com 4% de FBS), pelo menos 1 hr antes de adicionar a mistura de transfecção siRNA. </li><li> Prepare quatro tubos para a mistura de oligo transfecção siRNA. Em tubos A e C, adicionar 250 uL de Optimem soro reduzida e 4 ul de Lipofectamina RNAiMax, por 35 milímetros prato a ser transfectada. No tubo B, adicionar 250 mL de Optimem reduzido soro e 8 mL (160 pmol) de controle de conteúdo GC médio oligo (Controle siRNA), por 35 milímetros prato a ser transfectadas. Em tubo D, adicionar 250 mL de Optimem reduzido soro e 8 mL (160 pmol) de SMPD1 oligo (siASM), por 35 milímetros prato to ser transfectados. Incubar os tubos à temperatura ambiente durante 5 min. </li><li> Combinar os tubos A e B para formar o complexo de transfecção para o Controle de siRNA, e tubos de C e D para o ASM siRNA. Incubar as reacções à temperatura ambiente durante 20 min. </li><li> Adicionar as misturas de reacção a transfecção de controlo siRNA e ASM siRNA lentamente, gota a gota, para os respectivos pratos de fundo de 35 mm de vidro e incubar a 37 ° C / 5% de CO 2. Às 24 horas após a transfecção, aspirar delicadamente fora da mídia e substitua-o por meio de crescimento fresco DMEM. Para eficiente knockdown ASM, os pratos devem ser ainda incubada a 37 ° C / 5% de CO 2 durante cerca de 31 horas (total de 55 horas) antes de imagens em tempo real. </li></ol> 2. Preparação de Reagentes para Live Microscopia <ol><li> Prepara-se uma solução de toxina recombinante formador de poros estreptolisina O (SLO), a uma concentração de 100 ng / mL em PBS frio 1x sem Ca 2 + e Mg 2 +. A SLO utilizado nestes ensaios é um constitutivelmutante y activo cisteína que não requer agentes para reduzir a actividade, gentilmente fornecido pelo Dr. Rod Tweten, University of Oklahoma. A toxina foi expressa em E. coli BL21 DE3 e purificado sob condições nativas, como descrito previamente 16. </li><li> Preparar a solução de estoque FM1-43: Ressuspender FM1-43 em pó liofilizado em DMSO para criar uma solução de 25 mM. </li><li> Preparar a solução de A: Diluir a solução estoque FM1-43 em DMEM frio com Ca 2 + para uma concentração final de 4 | iM (1,5 ml são necessários por MatTek prato) e armazenar em gelo até ser necessária. </li><li> Prepare a Solução B: Diluir a solução estoque FM1-43 em DMEM frio, sem Ca 2 + e 10 mM de EGTA (Ca 2 +-DMEM livre) para uma concentração final de 4 | iM (1,5 ml necessários por MatTek prato) e armazenar em gelo até necessário. </li><li> Pré-quente (37 ° C) 1 ml de solução A e solução B, em tubos de Eppendorf. </li><li> Mantenha em soluções de gelo de DMEM com Ca 2 + e Cum DMEM 2 sem +. </li></ol> 3. Imagens de células vivas de FM1-43 Influx em SLO tratadas e células não tratadas <ol><li> Encher um recipiente metálico raso de tamanho médio com gelo moído, invertê-lo num recipiente de vidro grande com gelo, e adicionar gelo adicional deixando apenas a superfície do fundo do recipiente de metal exposto. Coloque uma toalha de papel molhado na parte inferior metal exposto para permitir a transferência, mesmo térmica. </li><li> Pegue um tratado com siRNA controle prato MatTek (Amostra 1) e coloque-a sobre o papel toalha molhada fria. Gentilmente aspirar fora todas as mídias e lavar 3x com frio Ca 2 + livre DMEM. Após a última lavagem, aspirar fora todas as mídias no prato. </li><li> Para imagem associada à célula FM1-43 em células sem SLO ferida (Exemplo 1) na presença de Ca2 +, adicionar 180 mL de solução fria B para a lamela de vidro do centro do prato de fundo de 35 mm de vidro e incubar durante 5 min em gelo (Tabela 1). </li&gt; <li> Coloque MatTek prato na plataforma de um microscópio confocal de aquecida a 37 ° C e equipado com uma câmara ambiental (o qual mantém a temperatura, a humidade e os níveis de CO 2). Encontre o campo de células que será fotografada olhando através de um 1,3 objetiva de 40X NA (Nikon). Se estiver disponível, ligue PerfectFocus ou um dispositivo semelhante para corrigir a deriva térmica. </li><li> Adicionar 1 ml de solução A pré-aquecido suavemente para o lado do prato de 35 mm e substituir tampa do compartimento do meio ambiente (Tabela 1). </li><li> Adquirir imagens para 4 min a 1 frame a cada 3 s usando software de imagem apropriado (Volocity no sistema de microscopia confocal disco giratório UltraView Vox Perkin Elmer). Excitação de FM1-43 é realizada com uma linha de laser 488 nm, utilizando um filtro dicróico com uma banda de passagem de 488 nm e de emissão de discriminação é conseguido através do uso de um filtro de emissão de 527 (W55). Níveis de potência do laser e sensibilidade da câmara são definidas para atingir exposições de imagem de 100 ms. </li><li> Repita os passos 3.2 a 3.6 para detectar FM1-43 em células de controlo tratadas com siRNA sem ferimento SLO (Amostra 2), na ausência de Ca2 +, excepto que devem ser adicionadas no passo 3.5 Solução B (Tabela 1). </li><li> Para medir a FM1-43 fluxo em células tratadas com siRNA de controlo feridos com SLO quer na presença (Exemplo 3) ou ausência de Ca2 + (Exemplo 4), adicionar 180 mL de solução fria B contendo SLO para a lamela de vidro do centro 35 mm prato fundo de vidro e incubar por 5 min no gelo como no passo 3.3. Repita os passos 3,2 por 3,6; ajustar adicionando ou Solução A Solução B ou no passo 3.5 (Tabela 1). </li><li> Para determinar o papel da ASM no reparo da membrana plasmática, repita os passos 3,2-3,6 usando células tratadas com siRNA ASM para todas as condições (amostras 5-8) (Tabela 1). </li><li> Para determinar o papel de esfingomielinase extracelular recombinante (SM) nacinética de reparação da membrana plasmática (reflectidos pela inibição em FM1-43 afluxo) (Exemplos 9-10), SM é adicionado (0-50 mU), quer da solução A ou solução B na etapa 3.5 e, em seguida, adicionado às células durante células vivas imagiologia de um volume total de 1 ml (Tabela 1). </li></ol> 4. Quantificação de FM1-43 em células Afluxo <ol><li> Após os filmes tenham sido adquiridos, medir a intensidade de fluorescência intracelular de FM1-43 usando o software de análise de imagem (Volocity Suite, PerkinElmer). Desenhar uma região de interesse na região citosólica de cada célula no campo e aplicar as ferramentas de software para determinar a FM1-43 intensidade média de fluorescência ao longo de todos os quadros de vídeo. </li><li> Para ajustar as variações na inicial FM1-43 coloração, os dados para cada vídeo é expresso como vezes de aumento na intensidade de fluorescência ao longo do tempo, e representada para cada condição diferente. O vezes de aumento na fluorescência de cada ponto de tempo individual porcélula é calculada como F T / F 0, onde T é F a fluorescência média no ponto de tempo específico, e F 0 é a fluorescência média em t = 0 (Figuras 1-2). </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Estudos anteriores sobre o prejuízo e membrana plasmática reparo mecânico contou com diversos mecanismos de células ferindo, desde soltando pérolas de vidro, micropipetting, corte, arranhão ou raspagem. A leitura de todos estes ensaios foi qualitativa do que quantitativa, e não dar informações precisas sobre a cinética do mecanismo de reparo. A capacidade das células para voltar a selar a sua membrana de plasma após estas formas de lesão foi medida através da presença ou ausência de marcadores adicionado...

Ele tem sido conhecido há várias décadas que o reparo da membrana plasmática após a lesão mecânica é um 1,2 Ca 2 + processo-dependente. Estudos posteriores mostraram que o influxo de Ca2 + através da ferida provoca um processo de vigorosa de exocitose de vesículas intracelulares no local da lesão, que é necessária para selar 3,4. A taxa de perda de um corante fluorescente carregados para o citoplasma das células foi usada para avaliar a velocidade de reparação, e concluiu que resselagem foi completada dentro de &lt;30 seg após a lesão 5. Dois modelos foram inicialmente propostos para explicar a exigência de exocitose no reparo da membrana plasmática: 1) o modelo de &quot;patch&quot;, que sugeriu que influxo de Ca2 + através da lesão desencadeia fusão inicialmente homotipica de vesículas intracelulares, formando um grande &quot;patch&quot; que faria em seguida, ser aplicado para a membrana plasmática para selar a ferida 6 e 2) o modelo de redução de tensão, que propôs que a membrana added por Ca 2 + dependente de exocitose na vizinhança da ferida iria reduzir a tensão da membrana plasmática, o que facilita a nova selagem da bicamada 7. O papel do Ca 2 +-exocitose desencadeado no reparo da membrana plasmática foi reforçada por estudos que mostram que os lisossomos contêm uma molécula de Ca 2 + sensor, sinaptotagmina VII, o que facilita a sua exocitose e reparo da membrana plasmática das células lesadas 8,9,10,11 . No entanto, a evidência adicional indicou que exocitose por si só não foi suficiente para promover a reparação da membrana plasmática. Além lágrimas mecânicas na membrana do plasma, uma forma comum de lesão celular é a permeabilização de toxinas formadoras de poros produzidos por bactérias ou células imunitárias 12,13 14,15. Ao contrário de lágrimas mecânicos, proteínas formadoras de poros se inserir na membrana plasmática formando uma poro revestido a proteína estável, que não pode ser fechado de novo, simplesmente por aplicação de uma membrana de &quot;remendo&quot; ou por reduçãoção de tensão de membrana. Curiosamente, os estudos revelaram que as células de mamíferos têm um mecanismo eficaz para reparar a sua membrana de plasma após a permeabilização com proteínas formadoras de poros, e esse processo também requer a presença de extracelular de Ca 2 + 12. Esta descoberta levantou a questão de saber se a remoção transmembranar poros da superfície da célula, também é um processo rápido, tal como observado com feridas mecânicas 5. Surpreendentemente, os nossos estudos recentes mostraram que a nova selagem de células permeabilizadas com toxinas formadoras de poros tem propriedades muito semelhantes às da reparação de feridas mecânicas: o processo requer extracelular de Ca 2 +, e é completado dentro de ~ 30 segundos. Investigando esse processo com mais detalhes, que recentemente descobriu que, além de Ca 2 + exocitose regulada de lisossomos, reparo da membrana plasmática envolve uma forma rápida de endocitose, que é desencadeada pela liberação da enzima esfingomielinase ácida lisossomal (ASM) e é essencial não só para poe remoção dos poros transmembranares, mas também para a reparação de feridas mecânicos 16. Para determinar a cinética de relacração celular após permeabilização com proteínas formadoras de poros, em nosso laboratório, adaptado de uma metodologia de imagens ao vivo que tinha sido usado anteriormente para avaliar a nova selagem de fibras musculares isoladas ferido a laser 17. Este ensaio baseia-se nas propriedades do corante lipofílico FM1-43, que se intercala estavelmente no folheto exterior de bicamadas lipídicas aumento na intensidade de fluorescência. Quando a bicamada da membrana do plasma é interrompido ganhos corante extracelular acesso às membranas intracelulares, fornecendo um ensaio sensível para detecção de lesões da membrana plasmática e reparar 18,16,19,20. Para adaptar este ensaio para a avaliação de resselagem célula após permeabilização com proteínas formadoras de poros, se as células pré-incubadas a 4 ° C com a toxina bacteriana estreptolisina O (SLO), que se liga ao colesterol da membrana 21. Célula Synchronouspermeabilização pode, então, ser facilmente conseguido movendo as células de gelo para aquecer meio de uma fase de microscópio aquecida, a qual ativa a oligomerização e as alterações na conformação que leva à formação de poros transmembranares. Uma vantagem desta abordagem, ao longo dos ensaios publicados anteriormente usando o ferimento a laser, é de que um maior número de células podem ser analisadas simultaneamente num campo microscópico, proporcionando uma melhor amostragem da população de células. Dadas as semelhanças entre o processo de mecanicistas resselagem celular observada após a lesão mecânica e permeabilização com toxinas formadoras de poros, o ensaio aqui descrito proporciona um método muito versátil e eficiente para factores envolvidos no processo fundamental de reparação da membrana plasmática de dissecação. Como exemplo, mostra-se que é possível utilizar este ensaio para identificar os passos de Ca 2 + dependente e independente do processo de reparação.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

Lesão da membrana plasmática é um evento freqüente e feridas têm de ser rapidamente reparado para garantir a sobrevivência celular. Influxo de Ca2 + é um evento chave de sinalização que desencadeia a reparação de feridas mecânicas na membrana de plasma no interior ~ 30 seg. Estudos recentes revelaram que as células de mamíferos também selar sua membrana plasmática após permeabilização com poros formando toxinas em um processo de 2 + dependentes de Ca que envolve a exocitose da esfingomielinase ácida enzima lisossomal seguido por poro endocitose. Aqui, nós descrevemos a metodologia utilizada para demonstrar que os fechos das células permeabilizadas pela toxina estreptolisina também é rápida e dependente de influxo de Ca2 +. O desenho do ensaio permite a sincronização do evento lesão e uma medição precisa da cinética da capacidade de células para restaurar a integridade da membrana plasmática por imagem e quantificar o grau pelo qual o corante liphophilic FM1-43 às membranas intracelulares. Este live als ensaioo permite uma avaliação da sensibilidade da capacidade de exogenamente adicionados factores solúveis, tais como a esf ingomielinase para inibir a FM1-43 afluxo, que reflecte a capacidade de células para reparar a sua membrana plasmática. Este ensaio permitiu demonstrar pela primeira vez que esfingomielinase actua a jusante de Ca 2 + dependente de exocitose, desde adição extracelular da enzima promove a re-selagem de células permeabilizadas, na ausência de Ca 2 +.

Baixas concentrações de reparo da membrana plasmática bacteriana esfingomielinase (SM) de resgate em células empobrecido em esfingomielinase ácida lisossomal. Usando o ensaio de imagiologia FM1-43, que mostraram previamente que as células deficientes para a enzima lisossomal ASM e expostas a SLO ferindo na presença de Ca2 + têm um defeito membrana plasmática é resgatado pela adição extracelular da enzima humana recombinante 19. Desde o ASM lisossomal humana tem...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

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

Imagens ao vivo de células expostas ao corante lipofílico FM1-43 permite a determinação precisa da cinética pelo qual as toxinas formadoras de poros são removidas a partir da membrana plasmática. Este é um ensaio sensível, que pode ser usado para avaliar os requisitos para Ca 2 +, esfingomielinase e outros factores de reparação da membrana plasmática.

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 ...

live-imaging-assay-for-assessing-roles-ca2-sphingomyelinase-repair

Research

JoVE Journal

Biology

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

11.6K visualizações.  University of Maryland.  Imagens ao vivo de células expostas ao corante lipofílico FM1-43 permite a determinação precisa da cinética pelo qual as toxinas formadoras de poros são removidas a partir da membrana plasmática. Este é um ensaio sensível, que pode ser usado para avaliar os requisitos para Ca 2 +, esfingomielinase e outros factores de reparação da membrana plasmática. 

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

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Elaboração de Drosophila Os embriões para Live-Imaging Usando o protocolo gota suspensa

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Biologia

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

Ressonância paramagnética eletrônica Micro-imagem das Espécies ao vivo para o Mapeamento de oxigênio

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Imagens ao vivo de cálcio celular Combinado com siRNA silenciamento gênico mediado Identifica Ca 2 + Canais de vazamento na membrana ER e seus mecanismos de regulação

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Um ensaio de explante para avaliar o comportamento celular do mesênquima craniana

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

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  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Imagens de células vivas dos primeiros autofagia Eventos: Omegasomes and Beyond

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

O tomate / GFP-FLP / FRT Method for Imaging ao vivo de Mosaico Adulto<em&gt; Drosophila</em&gt; As células fotorreceptoras

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...