The authors thank Mike Myles, Sam Saunders, and C.W. Elton at the ECU Multimedia &amp; Technology Services for providing assistance in video production. We acknowledge the grant support from North Carolina Biotechnology Center, Golfers against Cancer, Brody Brothers Endowment Fund, American Heart Association, and ECU/Vidant Cancer Research and Education Fund (L.V.Y. and M.J.R.).

1. Cultura Celular Wound Encerramento Assay <ol><li> Separar as células a partir da placa de cultura de tecido usando 0,25% de solução de tripsina-EDTA. Células de pellets em um tubo de 15 ml por centrifugação, aspirar o sobrenadante e as células em meios de cultura re-suspensão. Placa o número adequado de células em uma placa de 6 poços para 100% de confluência em 24 horas. Nota: Os testes podem ser necessários para determinar o tempo e o número de células para atingir 100% de confluência devido ao tipo de célula e a dimensão do bem a ser utilizado (por exemplo, 1 x 10 6 B16F10 células de melanoma são semeadas em um poço de uma 6 poços da placa, 24 horas antes da geração da ferida). Alternativamente, pode ser utilizado de 12 poços ou de 24 poços, placas. Nota: Se estiver disponível, as células podem ser semeadas em uma cultura de inserção (a partir de LLC Ibidi) em uma placa de cultura de tecidos tratados fazendo a ferida pré-fundido. Uma cultura-inserção pode proteger a superfície da placa de cultura de tecido de danos por a ponta da pipeta. Se uma Cultura-Insert é usado passo omitir1.2. </li><li> Em um ambiente estéril (tipicamente um capuz biossegurança) utilizar uma pipeta de ponta de 200 ul para pressionar firmemente contra o topo da placa de cultura de tecidos e fazer rapidamente uma ferida vertical descendente através da monocamada de células. Uma ponta de pipeta de tamanho diferente podem ser usadas para reduzir o tamanho da ferida, que é desejado. Não use força excessiva contra a placa de cultura de tecidos com a ponta da pipeta, pois pode danificar a superfície. Se a ferida é pré-fundido, este passo pode ser omitido. </li><li> Aspirar cuidadosamente a mídia e os restos celulares. Lentamente, adicionar meios de cultura suficiente contra a parede do poço para cobrir o fundo do poço e evitar separar células adicionais. Após a geração e inspeção da ferida uma visão inicial devem ser tomadas. Colocar a placa de cultura de tecidos numa incubadora à temperatura adequada e concentração de CO 2 (tipicamente 37 ° C e 5% CO 2). </li><li> Em vários momentos, por exemplo, a cada 3 horas, retire a placa da incubatou e colocá-lo sob um microscópio invertido para tirar uma foto instantâneo e para verificar o fechamento da ferida. Se microscopia de lapso de tempo está disponível, tirar uma foto a cada X número de minutos (normalmente começando com a cada 5 minutos) para qualquer duração de tempo em uma temperatura e CO 2 câmara controlada. Dependendo do tipo de célula, o tempo de fechamento da ferida pode variar. </li><li> Para analisar os resultados de imagens em fotografias, medir a distância de um lado da ferida para o outro usando uma barra de escala. Analisar e claramente presente o fechamento da ferida ao longo do tempo através de um gráfico de dispersão ou gráfico de barras, por exemplo, Figura 1. </li><li> Para analisar o lapso de tempo imagens, importar as fotos para o software ImageJ (disponível gratuitamente no http://rsb.info.nih.gov/ij/) para fazer um vídeo. Para fazer isso ImageJ aberto, clique em Arquivo, Importar e, em sequência de imagens. Localize o arquivo com as imagens; clique na primeira foto no arquivo. Salve vídeo, escolhendo Arquivo, Salvar como, e AVI. Células migratórias individuais podem ser observadas a characterize movimento direcional. Além disso, as características morfológicas, tais como formação lamellipodia e arrastando retração da borda podem ser estudados, bem como (Figura 2 e de vídeo suplementar). </li></ol> 2. Transwell migração celular e invasão de Ensaio <ol><li> Em um ambiente estéril (tipicamente um capuz biossegurança) separar as células da placa de cultura de tecido usando um tampão de dissociação não enzimática de células ou 0,25% de solução de tripsina-EDTA, as células por centrifugação, e aspirar os meios de comunicação existente deixando as células peletizadas. Re-suspender as células em meio de cultura celular livre de soro contendo 0,1% de BSA (albumina sérica bovina). Nota: Dependendo da linhagem de células a ser investigado 0,25% Tripsina-EDTA pode afectar a migração celular e invasão devido à clivagem de diversos receptores na superfície das células 17. Nota: o número de células por ml depende do tamanho das células. Podem ser necessários mais testes para descobrir a densidade de semeadura que provides os melhores resultados. Normalmente 1 x 10 6 células / ml é um bom ponto de partida para a maioria dos tipos de células ao usar a 24 poços transwell inserir. Existe uma gama de tamanhos de pastilhas transwell e o número de células adicionadas deve ser ajustado em conformidade para. </li><li> Placa de 100 ul de solução de células no topo da membrana de filtro em um transwell insert e incubar durante 10 minutos a 37 ° C e 5% de CO2 para permitir que as células se estabelecer. Nota: O tamanho específico dos poros da membrana Transwell utilizado é dependente do tamanho individual das células na amostra. Por exemplo, se as células forem muito pequenas que podem passar através dos poros, sem a necessidade de facilitar a migração ou se elas forem muito grandes não podem passar através dos poros. Existem várias inserções Transwell com tamanho de poro que variam de 3 mM, 5 mM, e 8 mm para os ensaios de migração celular. As inserções Transwell estão comercialmente disponíveis a partir de companhias tais como a Corning. Nota: O transwell migration ensaio pode ser facilmente modificado para realizar o ensaio de invasão celular. Para fazer isso, adicionar matriz extracelular (ECM) materiais na parte superior da membrana transwell e depois adicionar células no topo da ECM. Por exemplo, Matrigel é descongelado em gelo e liquefeito, e, em seguida, 30-50 mL de Matrigel é adicionada a um 24 bem transwell inserir e solidificado numa incubadora a 37 ° C durante 15-30 minutos de modo a formar uma fina camada de gel. Solução de células é adicionado no topo do revestimento de Matrigel para simular invasão através da matriz extracelular. Nota: Existem diferenças distintas entre a migração de células transwell e os ensaios de invasão celular transwell. O ensaio de migração celular transwell mede a capacidade quimiotática de células em direção a um quimio-atrativo. O ensaio de invasão celular transwell, no entanto, medidas, tanto a quimiotaxia de células ea invasão das células através da matriz extracelular, um processo que é comumente encontrada em metástase do câncer ou o desenvolvimento embrionário. </li><li> Com uma pipeta, muito carefully adicionar 600 ul do quimio-atractor desejado na parte inferior da câmara inferior de uma placa de 24 poços. Adicione o quimio-atrativo, sem mover a inserção transwell e evitar a geração de bolhas. Certifique-se de que o líquido quimio-atractor no poço inferior entra em contacto com a membrana na parte superior do poço, para formar um gradiente quimiotáctica. O tempo de incubação está dependente do tipo de célula e do quimio-atractor de ser utilizado. Nota: podem ser necessários mais testes para determinar o período de incubação. Nota: Para as células aderentes, as células que migraram vai anexar o outro lado da membrana de 1,8. A quantificação de células que migraram pode ser realizado seguindo os passos 2,4-2,8 (2,4-2,8 passos não necessitam de ser realizadas num ambiente estéril). Para as células não aderentes, as células migraram vai cair na mídia na câmara baixa. O número de células que migraram podem ser contadas usando um hemocitómetro ou citómetro de fluxo 5. </li><li> Remova a inserção da placa transwell. Use um aplicador com ponta de algodão quantas vezes for necessário para remover cuidadosamente a mídia e as células restantes que não migraram a partir do topo da membrana sem danificá-lo. </li><li> Adicionar 600-1.000 ul de etanol a 70% dentro de um poço de uma placa de 24 poços. Coloque a inserção transwell em etanol a 70% durante 10 minutos, para permitir a fixação das células. Retirar transwell insert a partir da placa de 24 poços e utilizar um aplicador com ponta de algodão para remover o etanol remanescente a partir da parte superior da membrana. Permitir que a membrana transwell para secar (tipicamente 10-15 minutos). </li><li> Adicionar 600-1.000 ul de 0,2% de violeta de cristal em um poço de uma placa de 24 poços e posicionar a membrana em que para a coloração. Incubar à temperatura ambiente durante 5-10 minutos. </li><li> Suavemente remover o violeta cristal a partir do topo da membrana com uma ponta de pipeta ou aplicador com ponta de algodão. Com muito cuidado, para evitar lavar as células fixas, mergulhe a membrana em água destilada como quantas vezes for necessário para remover o excesso viole cristalt. Permitir que a membrana Transwell para secar. </li><li> Vista sob um microscópio invertido e contar o número de células em diferentes campos de visão, para obter um valor médio de células que migraram através da membrana para a quimio-atractor e ligados no lado inferior da membrana (Figura 3). </li></ol>

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The authors declare that they have no competing financial interests.

A migração celular é um aspecto importante para estudar na pesquisa do câncer e também pode ser aplicado a estudos de cura de desenvolvimento, imunológicos e ferida. O ensaio de cultura de células de feridas e de fecho da migração celular e invasão transwell ensaios de revelar a informação detalhada do comportamento migratório das células e pode ser utilizado para investigar os mecanismos moleculares da migração celular 1,2,10,14. Nosso estudo utilizou estes testes de motilidade celular para d...

Motilidade é uma característica essencial de células vivas. A migração celular está envolvido na concepção de vida, o desenvolvimento embrionário, a resposta imunitária, e muitos processos patológicos, tais como a metástase do cancro e inflamação 1-9. Portanto, métodos para estudar o comportamento migratório das células são ferramentas de pesquisa muito útil para uma vasta gama de disciplinas em ciências biomédicas, biologia, bioengenharia, e áreas afins. O estudo da migração de células na pesquisa do câncer é de particular interesse como a principal causa de morte em pacientes com câncer está relacionado à progressão metastática. Para que o câncer se espalhar e disseminar por todo o corpo, as células cancerosas devem migrar e invadir através da matriz extracelular (ECM), intravasate em circulação no sangue, anexar a um local distante, e, finalmente, extravasar para formar focos distantes 1,10-12. Vários métodos biológicos podem ser utilizados para estudar estes eventos em detalhe. A cultura de células ferida fechado umd a migração e invasão transwell ensaios são amplamente utilizados na comunidade científica 1,10. Estes testes podem fornecer os dados necessários que possam permitir uma compreensão de como um tipo particular de célula podem migrar espontaneamente ou responder a uma quimioterapia atrativo e direcionalmente migrar em direção a ela. Vários fenótipos migradores foram descritos. As células podem migrar de uma forma única célula, como visto em mesenquimais ou movimento amebóide-like ou pelo movimento multicelular rotulado migração coletiva ou célula streaming de 13. O método de movimento usado em células móveis podem ser facilmente observado utilizando o ensaio de encerramento de feridas de cultura de células. Entre as várias formas de estudar a migração de células, o ensaio de encerramento de feridas de células é um dos mais simples. Este método é útil para determinar a capacidade de migração de massas de células inteiras. Quando mais um passo que pode ser usado para observar as características morfológicas da célula individual durante a migração 14. Após a análise do fechamento da ferida muitos fenótipos pode ser revelado. Medindo a distância fechado ao longo do tempo, quando comparado com um controle pode revelar alterações de migração específica ou um fenótipo migratório prejudicada que era desconhecida anteriormente. Além disso, a formação única lamellipodium celular, cauda retração, e movimento direcional pode dar pistas para o que pode ser prejudicada ou melhorada nas células de interesse 14. A migração transwell e ensaios de invasão pode ser utilizada para analisar a capacidade das células individuais para responder direcionalmente para vários quimio-atractores se são quimiocinas, factores de crescimento, lípidos, ou nucleótidos 4,5,8,15,16. Pode também avaliar a capacidade migratória diferencial devido à sobre-expressão de um receptor de 1,14. Estes ensaios também pode ser utilizado para identificar e caracterizar os reguladores-chave da migração de células tais como a família Rho de GTPases pequenas 2. Na sequência destes curto e de fácil acetestes veis, o modo de migração celular e a capacidade de uma célula para invadir numa matriz 3-D também pode ser determinada.

<table border="0" cellpadding="0" cellspacing="0" width="601">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">11995-073</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Fetal bovine serum (FBS)</td>
			<td style="width:133px;">Gemini</td>
			<td style="width:107px;">100106</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Bovine serum albumin (BSA)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:107px;">A4503-50G</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Trypsin EDTA 0.25%</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">25200-056</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Crystal violet</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:107px;">C0775-100G</td>
			<td style="width:193px;">Dissolved in water at 0.2%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Cell disassociation buffer</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">13151-014</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Cell culture incubator</td>
			<td style="width:133px;">Thermo Fisher Scientific</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # 3145</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">SterilGARD biosafety hood</td>
			<td style="width:133px;">The Baker Company, Inc.&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # VBM-600</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">EVOS Fl inverted microscope</td>
			<td style="width:133px;">Thermo Fisher Scientific</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # AMF-4302-US</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:169px;">Tissue culture plate</td>
			<td style="width:133px;">Becton Dickinson</td>
			<td style="width:107px;">353046</td>
			<td style="width:193px;">Catalog number varies depending on the type of culture plate</td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:169px;">Corning Transwell insert</td>
			<td rowspan="2" style="width:133px;">Fisher</td>
			<td rowspan="2" style="width:107px;">07-200-150</td>
			<td rowspan="2" style="width:193px;">Catalog number varies depending on the pore size of the membrane</td>
		</tr>
	</tbody>
</table>

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.

O ensaio de encerramento de feridas e no ensaio de migração de células Transwell aqui apresentados foram realizadas utilizando células de melanoma de ratinho B16F10 como um sistema modelo. No ensaio de encerramento de feridas, B16F10 células foram semeadas numa placa de 6 poços de cultura de tecidos e crescidas até 100% de confluência em 24 horas. Uma ferida de aproximadamente 700 m de largura foi gerada usando uma ponteira eo fechamento da ferida (migração de células) foi gravado usando a microscopia de laps...

Watch this Scientific Journal Video about In vitro Cell Migration and Invasion Assays at JoVE.com

In vitro Cell Migration and Invasion Assays

Comumente usado, são descritos métodos altamente acessíveis para analisar a migração celular e invasão in vitro. O primeiro método é o ensaio de encerramento de feridas de células que mede a mobilidade celular. O segundo método é o ensaio de migração e invasão transwell que avalia o quimiotáctica e capacidade invasiva de células.

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 ...

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In vitro Cell Migration and Invasion Assays

Unit 1

Cell Polarization and Migration

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137.2K visualizações.  East Carolina University.  Comumente usado, são descritos métodos altamente acessíveis para analisar a migração celular e invasão in vitro. O primeiro método é o ensaio de encerramento de feridas de células que mede a mobilidade celular. O segundo método é o ensaio de migração e invasão transwell que avalia o quimiotáctica e capacidade invasiva de células. 

Vídeo: In vitro Cell Migration and Invasion Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Ensaios Neurocircuit para Apreensões em mutantes de Epilepsia Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Biologia

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Um In vitro FluoroBlok Assay invasão tumoral

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Detecção rápida de Homogêneos Ensaios Biológicos Usando o sistema de modulação Biossensoriais Magnetic

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro Agregação ensaios utilizando hiperfosforilada Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

Métodos para a pele ferimento e Ensaios para Responses ferida no<em&gt; C. elegans</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Um sistema simplificado para a Avaliação celular Mechanosensing e Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

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 ...

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