Os autores reconhecem Programa criativo Inquérito da Universidade de Clemson e NSF CBET1254609 por fornecer financiamento para este projeto.

1. Produzir uma Câmara MicroChannel criar um gradiente de concentração <ol><li> Cortando uma peça de molde de acrílico <ol><li> Obter um pedaço de acrílico com a largura desejada. Normalmente, utilizam 1 placas de acrílico /-16 polegadas de espessura. folhas mais espessas são difíceis de cortar bem e muito finas folhas não têm a resistência mecânica, causando-lhes a quebrar ou deformar durante o processo. </li><li> Criar um ficheiro CAD com a forma desejada da peça de acrílico para produzir uma cavidade no interior do polidimetilsiloxano (PDMS). Ajustar a largura do canal e comprimento para atingir o desejado gradiente relevantes para protocolos experimentais individuais. <ol><li> Aqui, utilizar uma peça de acrílico com as seguintes dimensões: dois quadrados (0,254 cm x 0,254 centímetros) ligada por um canal de largura 0,127 cm (0,254 cm de comprimento) (Figura 1). </li></ol></li><li> Importar o arquivo CAD para dentro do aparelho de corte a laser e coloque a peça de acrílico em um cortador de laser de acordo com a manuo protocolo do cante. NOTA: Como alternativa, 3D-imprimir a peça a partir do desenho CAD. A vantagem deste método é que os materiais alternativos podem ser usadas para o molde; No entanto, a resolução e a reprodutibilidade pode ser diminuída com impressão em 3D em comparação com o corte a laser. </li></ol></li></ol><img alt="figura 1" src="/files/ftp_upload/55264/55264fig1.jpg" /> Figura 1: (CAD) Representação Computer-Aided Design de microcanais Câmara. Esta imagem descreve o desenho CAD da inserção acrílico necessário para criar a câmara de microcanais. 1 A) Vista de cima do desenho CAD, 1B) Vista lateral do desenho CAD com dimensões em polegadas, 1C) Vista superior de desenho CAD com dimensões em polegadas <a href="http://ecsource.jove.com/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <ol start="2"><li&gt; Verter o polidimetilsiloxano (PDMS) <ol><li> Em um barco de pesagem, misturar 10 partes em peso de base de elastómero comercial (por exemplo, Sylgard 184) com 1 parte em peso de um elastómero comercial Agente de cura (por exemplo, Sylgard 194). Tipicamente, misturar 10 g de Elastómero de base para 1 g de Elastómero Agente de cura. </li><li> Agita-se para combinar a base e o agente de cura com uma micropipeta durante 5 min. </li><li> Defina-se um sino de vácuo e coloque o barco pesar com PDMS no vácuo durante 30 min para remover as bolhas de ar aprisionadas. </li><li> Desligue o vácuo e remover o barco pesa. Despeje o PDMS em cima do acrílico cut-out em uma pequena placa de Petri. Certifique-se de que os PDMS cobre completamente a inserção. </li><li> Permitir que o PDMS a curar durante a noite (pelo menos 18 h) à temperatura ambiente. </li></ol></li><li> Removendo a inserção de PDMS <ol><li> Após a cura do PDMS, remova a inserção cortando cuidadosamente as PDMS ao redor do pedaço de acrílico com um bisturi. </li></ol&gt; </li></ol> 2. plaqueamento das células em uma Câmara MicroChannel <ol><li> Esterilizar a câmara de células de plaqueamento. NOTA: Enquanto PDMS pode ser esterilizado usando óxido de etileno ou outras técnicas, um tratamento rápido de UV é rápido, barato, e suficiente para estudos de culturas celulares. A duração do tratamento UV curto não prejudicar a estrutura mecânica ou a integridade da peça de PDMS. <ol><li> Em um armário de cultura celular padrão, cobrir completamente a câmara com 70% de etanol. </li><li> Coloque a placa de Petri na capela de fluxo laminar com as tampas fora. </li><li> Feche a tampa e ligar a luz UV. Expor à luz UV durante 1-2 h. </li><li> Abra a câmara de fluxo laminar e aguarde 15 minutos para restabelecer o fluxo. </li><li> Remover o etanol a 70%. </li><li> Lavam-se as câmaras duas vezes com solução salina estéril tamponada com fosfato (PBS). Use 1 ml de PBS para cada lavagem. Remover PBS e permitir câmaras para secar durante a noite com as tampas fora. NOTA: Como alternativa, em vezda utilização de uma câmara de fluxo laminar, use uma caixa de câmara de UV para esterilização se estiver disponível. Adicione uma caixa de UV colocando uma fonte de luz UV na parte de trás de uma caixa de cartão revestida com folha de alumínio. A folha de alumínio reflecte a luz para permitir a iluminação uniforme da amostra. </li></ol></li><li> Chapeamento de células em câmara <ol><li> Contagem de células utilizando azul tripano e um hemocitómetro. <ol><li> Adiciona-se 100 uL da suspensão de células de 400 uL de 0,4% de azul de Tripano e misturar suavemente. Adicionar 100 ul desta solução para o hemocitómetro preenchendo suavemente ambas as câmaras sob a lamela de vidro. </li><li> Use um microscópio com uma objetiva de 10X para se concentrar na grelha no hemocitômetro. Use um contador de contagem mão para contar as células não coradas ao vivo dentro de um conjunto de 16 quadrados no hemocitômetro. </li><li> Contagem de células em todas as 4 séries de 16 quadrados. Tome a contagem média de células a partir dos 4 conjuntos de 16 quadrados e multiplicar por 5x10 4. Este número final é o number de células / ml na suspensão de células. </li></ol></li><li> Adicionar 2500 células a uma cavidade em cada uma das câmaras. Isto traduz-se para uma densidade quase confluentes de células de ~ 30000 / cm 2. </li><li> Permitir que as células para se sentar na câmara durante 60 minutos antes de adicionar mídia (de Eagle Modificado por Dulbecco Médio, 10% de soro fetal bovino, 1% de penicilina-estreptomicina). </li></ol></li></ol> 3. Blocos de agarose Dextran Soaked <ol><li> Criando um bloco de agarose <ol><li> Pour 3% de agarose em molde 3D impressa (2 mm x 2 mm x 2 mm). </li><li> Permitir que a agarose solidificar em uma câmara de vácuo durante 30 min para remover quaisquer bolhas. </li><li> Remover o bloco de agarose a partir do molde. NOTA: Como alternativa, derrame 3% de agarose em pequena placa de Petri com uma espessura de 2 mm. Corte um bloco de 2 mm x 2 mm x 2 mm, utilizando um bisturi ou outra ferramenta de corte. Este não é tão preciso como o sistema de molde mas pode ser um pouco mais fáceis de fazer. </li></ol></li><li> completamente submerge o bloco de agarose numa solução com a concentração desejada do factor quimiotáctico. Para avaliar a formação de gradiente de concentração, lavar a câmara pela primeira vez com dextrano fluorescente. A concentração e peso molecular do dextrano deve coincidir com a de um factor solúvel ou droga de interesse. </li><li> Mergulhe a noite bloco de agarose. </li></ol> 4. Time-lapse of a difusão Dextran para avaliar o gradiente de concentração Solúvel Fator <ol><li> Coloque o bloco de agarose dextrano embebido em um pequeno poço quadrado da câmara de microcanais. </li><li> Coloque a câmara de microcanais em um sistema de imagens de células ou utilizar outro microscópio de fluorescência com uma capacidade de lapso de tempo. Comece o lapso de tempo de acordo com as instruções do fabricante. Nota: Os resultados apresentados são tomadas com filtro GFP, 50% de luz, 4/10 pH, e uma ampliação de 4X. </li></ol> 5. Time-lapse do crescimento celular <ol><li> células placa na câmara de microcanais em uma extremidade docâmara. Aqui, usar fibroblastos 3T3. Adicionar 2500 células a uma cavidade na câmara de microcanais. Contagem de células como descrito em 2.2.1. <ol><li> Permitir que as células para se sentar na câmara durante 60 minutos antes de adicionar mídia (de Eagle Modificado por Dulbecco Médio, 10% de soro fetal bovino, 1% de penicilina-estreptomicina). Manter a câmara de microcanais com células plaqueadas a 37 ° C em 5% de CO 2. A migração celular através do canal pode ser vista com um microscópio. </li><li> Adicionar um marcador ou utilização de um defeito microscópico na câmara como um marcador, a fim de servir como um ponto de partida para quantificar a distância que a célula move-se. </li></ol></li><li> Estabelecer um gradiente por colocação de um gel de agarose (8 mm3 bloco) que contém o factor de crescimento concentrado, quimio-atractor, droga, ou outro factor a ser ensaiado (aqui, 40% de FBS está a ser utilizada como um exemplo), a uma extremidade do microcanal câmara. </li><li> Tome imagens de lapso de tempo de frente da célula em movimento. Aqui, os resultados mostram imagens da célula front que foram tomadas a cada 12 h, utilizando um sistema de imagem. </li><li> Use imagens de frente da célula para quantificar a taxa de crescimento celular. Calcula-se a taxa de crescimento utilizando primeiro a função polyfit MATLAB (roipoly) para criar uma máscara poligonal definido pela frente da célula, as paredes do canal, e o marcador de referência. As dimensões desta máscara se obter a distância de migração da frente a partir do qual a velocidade média frente célula é calculada. Outros estudos têm utilizado um método semelhante 8. NOTA: Comparar a borda da frente de crescimento da célula em cada quadro para a concentração do factor solúvel naquela mesma distância. O gradiente de concentração factor solúvel é calculado utilizando um modelo de difusão 1D aproximação. </li><li> Tome imagens fluorescentes das células utilizando faloidina e DAPI coloração. <ol><li> Fix células antes da coloração com faloidina. <ol><li> Quente paraformaldeído a 4% a 37 ° C. </li><li> Adicionar suficiente paraformaldeído a 4% para cobrir as células. Manter as células naparaformaldeído durante 10 min. </li><li> Lavar duas vezes com PBS durante 15 min de cada vez. Use PBS suficiente para cobrir as células. </li></ol></li><li> Remover PBS a partir de células e adicionar solução de permeabilização suficiente (0,1% de Triton X-100 em PBS) para cobrir as células. Deixar solução durante 10 min. </li><li> Lavar duas vezes com PBS durante 5 min de cada vez. Use PBS suficiente para cobrir as células. </li><li> Remover PBS a partir de células e adicionar solução (1% de albumina de soro de bovino em PBS) de bloqueio durante 20 minutos. </li><li> Remoção da solução de bloqueio e lavar 2 vezes com PBS durante 5 min de cada vez. Use PBS suficiente para cobrir as células. </li><li> A partir deste ponto, a proteger as células da luz com folha de alumínio, quando não estiver sendo usado. Remover PBS a partir de células e adicionar 488 faloidina diluídas a 1:50 em PBS durante 1 h. </li><li> Lavar 2 vezes em PBS durante 5 minutos para cada lavagem. Use PBS suficiente para cobrir as células. Em seguida, retire PBS a partir de células. </li><li> Adicionar DAPI (4 &#39;, 6-diamidino-2-fenilindol) diluído 1: 1000 em PBS às células durante 15 min. </li><li> Remover DAPI elavar as células duas vezes com PBS durante 5 minutos para cada lavagem. </li><li> Remover última lavagem e adicione o suficiente PBS para cobrir as células. As células podem agora ser visualizados com um microscópio de fluorescência. </li></ol></li></ol>

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(69), (2012).</li></ol>

Os autores não têm nada a revelar.

A nossa câmara de microcanal pode ser utilizado para uma variedade de fins, incluindo a determinação da taxa de migração celular em resposta a factores de crescimento e quimioatractores e medindo a taxa de difusão de um fármaco a partir de uma matriz de polímero. É possível utilizar a câmara de microcanais para crescer as células e colocar um quimioatractor numa extremidade da câmara. As células crescem em resposta ao quimioatraente, e a migração de células pode ser quantificada tomando imagens de lapso...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
		</tr>
		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
		</tr>
	</tbody>
</table>

a customizable chamber for measuring cell migration

A migração celular é uma parte vital da resposta imune, crescimento e cicatrização de feridas. A migração celular é um processo complexo que envolve interacções entre células, a matriz extracelular e factores químicos solúveis e não solúveis (por exemplo, quimioatractores). Métodos padrão para medir a migração de células, tais como o ensaio de câmara de Boyden, trabalho por contagem de células em ambos os lados de uma divisória. Estas técnicas são fáceis de utilizar; no entanto, eles oferecem pouca modificação geométrica para diferentes aplicações. Em contraste, os dispositivos microfluidicos podem ser utilizados para observar a migração de células com gradientes de concentração personalização de factores solúveis 1, 2. No entanto, os métodos para fazer os ensaios com base microfluidos pode ser difícil de aprender. Aqui, nós descrevemos um método fácil para criar câmaras de cultura de células para medir a migração celular em resposta a gradientes de concentração química. Nossos mi celularesmétodo câmara gração pode criar diferentes gradientes de concentração linear de modo a estudar a migração de células para uma variedade de aplicações. Este método é relativamente fácil de usar e é normalmente realizada por alunos de graduação. A câmara de microcanais foi criado por colocação de uma inserção de acrílico na forma da câmara de microcanais finais bem em uma placa de Petri. Depois disso, o poli (dimetilsiloxano) (PDMS) foi deitada no topo da inserção. O PDMS foi deixada endurecer e, em seguida, a inserção foi removida. Isto permitiu a criação de cavidades em qualquer forma ou tamanho desejado. As células podem ser posteriormente adicionado à câmara de microcanais, e agentes solúveis podem ser adicionados a um dos poços por imersão de um bloco de agarose, o agente desejado. O bloco de agarose é adicionado a um dos poços, e as imagens de lapso de tempo pode ser feita da câmara de microcanal, a fim de quantificar a migração celular. Variações deste método pode ser feita para uma dada aplicação, tornando este método highly personalizável.

A Figura 2 mostra o movimento da parte dianteira da célula através do canal em resposta a um gradiente de soro fetal de bovino colocado na extremidade oposta do canal, de onde as células são plaqueadas. A parte dianteira da célula é mostrada em 48 h (Figura 2A), 72 h (Figura 2B), 96 h (Figura 2C), e 120 h (Figura 2D) pós chapeamento. O movimento da frente de celular foi rastreado com estas imagens...

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A Customizable Chamber for Measuring Cell Migration

Este protocolo detalha um método personalizada para medir a migração celular em resposta a quimioatractores, que também podem ser utilizados para determinar a taxa de difusão de um fármaco a partir de uma matriz de polímero.

A Câmara customizáveis ​​para a migração celular de medição

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

a-customizable-chamber-for-measuring-cell-migration

Research

JoVE Journal

Biology

10.5K visualizações.  Clemson University.  Este protocolo detalha um método personalizada para medir a migração celular em resposta a quimioatractores, que também podem ser utilizados para determinar a taxa de difusão de um fármaco a partir de uma matriz de polímero. 

Vídeo: A Customizable Chamber for Measuring Cell Migration

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Medição Exocitose em Neurônios Usando Labeling FM

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Biologia

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

Estimulação cerebral Slice Usando uma Rede microfluídicos e da Câmara de Perfusão padrão

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Imagens ao vivo da migração das células da glia no Disco Drosophila Eye Imaginal

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Medir a rigidez à flexão de células bacterianas Usando uma armadilha óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Um ensaio para medir a atividade do Escherichia coli Induzível Lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital da Microcirculação no músculo Rato Cremaster de Análise de Migração Stem Peripheral celular

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Microinjeção de uma única célula para análise da comunicação celular

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Na migração celular in vitro e invasão Ensaios

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Filamentos intermediários e microtúbulos com microscopia 2-dimensional direta estocástico reconstrução óptico de imagem

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

A Câmara customizáveis ​​para a migração celular de medição

A Câmara customizáveis para a migração celular de medição