Agradecemos Laurent Limozin, Pierre Dillard e Astrid Wahl para continuar discussões frutíferas sobre aplicações celulares. Agradecemos também Frederic Bedu de instalação de sala limpa PLANETE por sua ajuda com as observações SEM. Este trabalho foi parcialmente financiado pelo Conselho Europeu de Investigação através da concessão No. 307.104 FP / 2007-2013 / ERC.

1. A limpeza dos vidros Capa-slides e câmaras de observação <ol><li> Organizar as coberturas de lâminas de vidro sobre um tabuleiro multi-deslizante feito de um material inerte tal como o politetrafluoroetileno (PTFE). </li><li> Mergulha-se o tabuleiro com lâminas e a câmara de observação numa solução de agente tensioactivo (qualquer produto recomendado para a limpeza de cuvetes de quartzo é apropriado). </li><li> Utilizando um banho de ultra-sons, ultra-sonicado na solução de surfactante durante 30 min à temperatura ambiente (entre 20 e 30 ° C). </li><li> Lavar 5 vezes com água ultrapura (18,2 MΩ.cm, 0,059 S / cm). </li><li> Ultra-sonicado em solução surfactante durante 30 min à temperatura ambiente (entre 20 e 30 ° C). </li><li> Lavagem de 10 vezes com água ultrapura. </li><li> Repita os passos 1.5 e 1.6, duas vezes. </li><li> Armazenar em água à temperatura ambiente (entre 20 e 30 ° C) durante até uma semana. </li></ol> 2. Fabricação de proteína Nano-pontos <ol><li> Deposição de contas <ol><li> deposit a suspensão de contas de sílica (2% v / v, 70 mL) gota a gota em um-corrediça de cobertura (24 x 24 mm, espessura de 170 um), realizada com uma inclinação de 15 graus. </li><li> Deixe a suspensão espalhada durante cerca de 1 min, enquanto que lança o vidro de 90 ° a cada 15 s. </li><li> Permitir que o líquido se evaporar sob condições ambiente. </li><li> Armazenar em um vidro limpo ou câmara de PTFE em condições ambientais durante até 2 dias. </li></ol></li><li> Deposição de alumínio <ol><li> Colocar as lâminas (preparada na secção 2.1) dentro de uma frequência de rádio de pulverização catódica equipamento (RF) magnetrão 8, sobre uma mesa rotativa a uma distância de cerca de 105 mm a partir de uma lata de alumínio (99%): silício (1%) alvo. </li><li> Bombeiam para baixo a câmara de deposição de 2,6 x 10 -4 Pa através de uma bomba turbomolecular. Esta etapa é importante para a remoção de possíveis impurezas gasosas. </li><li> Introduzir puro atmosfera de Árgon (5N, 99,999%) com um fluxo de 10 sccm a uma pressão de 0,8 Pa (6,6 mTorr). <br/&gt; NOTA: O fluxo de árgon de entrada é controlado por um controlador de fluxo de massa e a pressão do processo é monitorizado utilizando um medidor de pressão. O fluxo de gás árgon é contínua e é controlado por um equilíbrio entre a entrada e válvulas de abertura de saída. </li><li> Ligue o gerador de energia RF. NOTA: Aqui, foi utilizada uma gama típica de 400-600 W de energia de radiofrequência a 13,56 MHz de frequência. O gerador de RF é utilizada com uma Rede de adaptao de um modo de acoplamento do plasma capacitiva. A energia reflectida é monitorado e minimizado utilizando a adaptação da rede. </li><li> Após o plasma é estabilizado, de descarga eléctrica durante dois minutos, mantendo o obturador fechado para remover possíveis impurezas da superfície do alvo. </li><li> Abrir o obturador e permitir a pulverização continue durante 60 min a depositar alumínio sobre a lâmina de vidro até uma espessura de 200 nm. </li><li> Cortar o fluxo de árgon, fechar a válvula de gaveta para isolar a bomba turbomolecular a partir da câmara de deposição e ventilar a câmara com n limpoitrogen para obter a pressão ambiente. Recuperar as lâminas revestidas com alumínio. Armazenar por até um mês em uma caixa de vidro ou PTFE limpo e hermeticamente fechado em condições ambientais. </li></ol></li><li> de deposição de vapor de organossilano <ol><li> Imergir a lâmina revestida de alumínio preparado no passo 2.2.6 em água ultrapura, à temperatura ambiente e ultra-sonicação durante 30 s num banho de ultra-sons (50 W, 50/60 Hz, entre 20 e 30 ° C). </li><li> Depósito de 0,5 mL de (3-aminopropil) -triethoxysilane (APTES) na parte inferior de um exsicador. CUIDADO: APTES é um organossilano, que evapora facilmente e é tóxico. APTES deve ser manuseado apenas sob um fluxo-capuz e com luvas. </li><li> Coloque as lâminas de vidro (preparadas em 2.3.1) em uma grade de cerâmica e coloque dentro do secador. </li><li> Ligue o exsicador para uma bomba de membrana e na potência máxima durante 30 min para gerar um vácuo baixo. </li><li> Fechar a válvula do exsicador e desligar a bomba. </li><li> Aquecer o secadora cerca de 50 ° C durante 1 h. </li><li> Abra o exsicador e recolher os slides. </li><li> Transferir para outro exsicador limpo para armazenamento de até 48 horas à temperatura ambiente (20 a 30 ° C). </li></ol></li><li> Deposição de primeira camada de proteína - albumina de soro bovino marcada com biotina (BSA-biotina) <ol><li> Coloque uma das lâminas preparadas na seção 2.3 sobre um suporte de PTFE. </li><li> Depósito 1 mL de 25 ug / mL de BSA-biotina dissolvida em tampão fosfato salino (PBS). Deixar durante 30 minutos à temperatura ambiente (20 a 30 ° C). </li><li> Enxaguar 10 vezes com PBS. Armazenar a amostra durante até 24 h a 4 ° C para longe da fonte de luz. </li></ol></li><li> Remoção de máscara de alumínio <ol><li> Incubar as lâminas numa solução de NaOH em PBS (preparado por adição gota a gota de NaOH 1 M a cerca de 100 mL de PBS para se obter pH≈12) durante a noite à temperatura ambiente (entre 20 e 30 ° C). CUIDADO. NaOH é corrosivo e deve ser manuseado com luvas. </li><li> Rinse10 vezes em água ultrapura. </li></ol></li></ol> 3. A deposição de Compatível Lipid Bilayer (SLB) <ol><li> Limpeza da calha Langmuir <ol><li> Remover a água no compartimento de PTFE da calha de Langmuir utilizando uma bomba. </li><li> Limpo com uma toalha descartável não tecido que não solte fiapos embebido em clorofórmio. CUIDADO. O clorofórmio é tóxico e deve ser manipulada numa área bem ventilada, com uma máscara (ou sob um fluxo-capa) e com luvas apropriadas. </li><li> Limpos 4 vezes com água ultrapura morna (40-50 ° C). </li><li> Limpas pelo menos 6 vezes com água ultrapura frio. </li></ol></li><li> A deposição da primeira camada de lípido <ol><li> Coloque bandejas de PTFE no recinto de PTFE da calha de Langmuir e depois enchê-lo com água ultrapura. </li><li> Usar o software de controlo do aparelho de Langmuir para definir a pressão medida a 0 mN / m. </li><li> Usar uma seringa estanque a gases de vidro / metal a depositar 30 uL de uma solução de lípidos de 1 mg / mL (1, 2-dioleoil-sn-glicero-3-fosfocolina (DOPC) em clorofórmio) sobre a superfície da água. O clorofórmio e evapora-se as moléculas de lípidos formam espontaneamente uma monocamada. </li><li> Usar o software de controlo para comprimir a monocamada de lípidos, fechando a barreira de PTFE até que a pressão desejada (27 mN / m para DOPC) é alcançado. </li><li> Usar o software de controlo para mergulhar a lâmina de vidro preparado na secção 2.5.2 para dentro do invólucro de PTFE utilizando uma braçadeira motorizado. </li><li> Segurar a corrediça, na braçadeira, perpendicular à interface ar-água. Usar o software de controlo para elevá-lo lentamente (15 mm / min) através da interface, enquanto se mantém uma pressão constante a 27 mN / m. </li><li> Lugar num ambiente seco, quer para uso imediato ou para armazenamento até 24 horas à temperatura ambiente (entre 20 e 30 ° C). </li></ol></li><li> A deposição da segunda camada lipídica <ol><li> Manter a pressão de superfície na tina de Langmuir no valor desejado de 27 mN / m. Comprimir usando osoftware do aparelho de Langmuir e / ou controlo de adicionar uma pequena quantidade de lípido para alcançar a pressão desejada. </li><li> Colocar as lâminas de vidro que transportam a monocamada de lípidos, preparada na secção 3.2.6, na horizontal sobre a superfície da água. Assegure-se que cada lâmina está a flutuar por cima de um tabuleiro de PTFE. </li><li> Empurrar as lâminas de vidro para baixo, uma lâmina de cada vez, para a sua bandeja de PTFE correspondente utilizando PTFE ou metal pinça de tal modo que eles são imersos na água. Evite inclinar os slides enquanto empurra. </li><li> Use PTFE ou metal pinças para transferir as bandejas de PTFE contendo os deslizamentos em um cristalizador preenchido com água ultrapura certificando-se de que as lâminas não são expostas ao ar. </li><li> Use PTFE ou metal pinças para transferir, durante o trabalho debaixo de água, uma bicamada revestido lâmina de vidro para uma câmara de observação. NOTA: A câmara de observação é colocado debaixo de água no interior do cristalizador. A câmara de observação é feito sob medida e consiste de um anel de PTFE com um uma junta de borrachainvólucros de aço d, que podem ser montados debaixo de água para fazer uma câmara estanque à água, cuja superfície inferior é feita a partir da lâmina de vidro revestida previamente com a bicamada lipídica. </li><li> Fechar a câmara, continuando a trabalhar com água e assegurando que cerca de 1 mL de água é preso no interior da câmara. </li><li> Levar a câmara montada fora da água. Certifique-se de que ele é livre à prova de água e vazamento. </li><li> Substituir o 1 ml de água ultrapura presente na câmara de observação com PBS por adição e remoção de 500 ul de PBS 10 vezes. Assegurar que a câmara nunca é desprovida de líquido. </li></ol></li><li> etapa de bloqueio SLB <ol><li> Introduzir 100 ug / mL de albumina de soro bovino (BSA) na câmara de observação contendo a corrediça preparada na secção 3.3.6 e incubar durante 30 min à temperatura ambiente (entre 20 e 30 ° C). </li><li> Lavar a camada dupla por eliminação e adição de 500 ul de PBS 10 vezes. A bicamada pode ser mantido 24 horas a 4 ° C. </li></ol></li></ol> 4. A funcionalização com ligantes <ol><li> Adicionar fluorescente ou não fluorescente neutravidina (NAV, uma versão desglicosilada de avidina não carregado a pH 7) a 2 ug / mL para a câmara contendo a corrediça preparada na secção 3.4.2, durante 30 min à temperatura ambiente (entre 20 e 30 ° C). </li><li> Lavar por adição e remoção de 500 ul de PBS 10 vezes. </li><li> Adicionar biotinilado anti-CD3 a 2 ug / mL para a câmara que contém a corrediça preparada na secção 3.4.2. Para dupla funcionalização do SLB e os pontos, adicionar Fc-ICAM1 His-tag, a 5 ug / ml (aqui, a bicamada é feita de DOPC + 1% de ácido nitrilotriacético (NTA) lípidos). Deixar durante 30 minutos à temperatura ambiente (entre 20 e 30 ° C). </li><li> Lavar por adição e remoção de 500 ul de PBS 10 vezes </li><li> Substituir o PBS com meio celular (PBS + BSA a 0,1%) por adição e remoção de 500 mL do meio de células 10 vezes. </li><li> Deixar 200 uL de meio de células no chamber com a corrediça. </li><li> Coloque a câmara durante 10 minutos a 37 ° C antes da adição de células. </li></ol> 5. Célula de deposição (ver referência 7 para detalhes) <ol><li> depositar cuidadosamente 400 ul da suspensão de células para dentro da câmara. Incubar as culas durante 30 min a 37 ° C. </li><li> Fixar a célula com 2% de paraformaldeído (PFA) durante 15 min a 37 ° C. Substituir o PFA com PBS por remoção repetida e a adição de 500 uL de PBS. </li></ol> 6. Observação <ol><li> Proteo-lipídico Nano-padrão e SLB fluidez <ol><li> Usar um microscópio de fluorescência para a imagem do padrão de proteínas no modo de epi-fluorescência usando comprimento de onda apropriado iluminação (639 nm) e cubos de filtro (por exemplo, aqui, EX TBP 483 + 564 + 642; BS TFT 506 + 582 + 659; EM TBP 526 + 601 688). NOTA: A intensidade do sinal pode ser quantificada para estimar a quantidade de proteína dentro e fora do ponto. Usar um comprimento de onda apropriado iluminação (330 nm) e fcubo ILTER a imagem da bicamada (BP 365/12, FT 395, LP 397), que aparece brilhante com buracos escuros. </li><li> Use foto contínuo de branqueamento (CEC) técnica 13, 15 para medir a constante de difusão de lípidos de traçador na bicamada. Esta observação é, de preferência feito apenas após a deposição SLB. NOTA: De modo a quantificar a constante de difusão, dois parâmetros são medidos durante o processo de CEC: o tempo de branqueamento específica do corante (tenso) e o comprimento de decaimento do perfil fluorescente do halo formado em torno da área branqueada (taud). O primeiro é obtido através da representação gráfica da evolução no tempo da intensidade de fluorescência no centro do campo iluminado durante o processo de CPB. A segunda é obtido por traçado do perfil de intensidade na borda da zona iluminada (média de mais de 12 linhas e 5 depois as imagens em estado estacionário é atingido). As curvas são montados para obter tenso e taud. A constante de difusão é dada por tauD 2 / tenso (D = <img alt="Equação" src="/files/ftp_upload/55060/55060eq1.jpg" /> ). </li></ol></li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+mobility+modulates+immunological+synapse+formation+and+T+cell+activation.">Ligand mobility modulates immunological synapse formation and T cell activation.</a> PloS One. 7 (2), e32398 (2012).</li><li>Yu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+integrin+binding+to+Arg-Gly-Asp+peptide+activates+actin+polymerization+and+contractile+movement+that+stimulates+outward+translocation.">Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.</a> Proc Natl Acad Sci U S A. 108 (51), 20585-20590 (2011).</li></ol>

Os autores não têm nada a revelar.

Os passos críticos dentro do protocolo descrito acima estão relacionadas com a formação da proteína nano-pontos ou a parte de trás de enchimento do espaço em torno dos pontos por uma bicamada lipídica suportado. O primeiro passo crítico com respeito a proteína nano-pontos é a preparação do grânulo-máscara. A limpeza da tampa a corrediça é crítica. As lâminas têm de ser limpas, quer com uma solução de detergente que é recomendado para a limpeza de cuvetes de quartzo, ou com plasma de oxigénio. Out...

<html> A adesão celular tem lugar através de moléculas de adesão de células especializadas (CAMs), proteínas presentes na membrana de células que são capazes de se ligar a sua contraparte na matriz extra celular ou noutra célula. Em culas aderentes, a maioria das moléculas de adesão incluindo a integrina ubíqua e caderina, encontram-se na forma de agregados 1. A interacção dos linfócitos T (células T) com as células apresentadoras de antígenos (APCs) fornece uma ilustração particularmente notável da importância dos aglomerados de receptores formados na interface entre as duas células - muitas vezes chamada uma sinapse imunológica. Após a formação do primeiro contacto com os receptores de células APC, T (TCRs) sobre a superfície dos agregados de escala forma célula micron T que servem de sinalização plataformas 2, 3, 4, e, eventualmente, são centralizados para formar um agrupamento supramolecular central maior (cSMAC )lass = &quot;xref&quot;&gt; 5, 6, 7. Recentemente, foi mostrado que no lado do APC, os ligandos de TCR também estão agrupados 8. No contexto de células T-APC interacção, a implementação de sistemas híbridos, onde o APC é mimetizado por uma superfície artificial funcionalizadas com proteínas relevantes, avançou significativamente a nossa compreensão da interface sináptica 2, 3, 4, 5, 6, 7 . Neste contexto, é altamente relevante para desenhar superfícies miméticos APC que capturam um ou mais aspectos da célula alvo. Por exemplo, se os ligandos são enxertados em bicamadas lipídicas com suporte, que pode difundir-se no plano da bicamada, mimetizar a situação na superfície da APC e ao mesmo tempo permitir que a formação dacSMAC 6, 7. Da mesma forma, os aglomerados sobre o APC foram imitada através da criação de ilhas de ligandos num mar de polímeros 9, 10, 11, 12, 13, 14. No entanto, estas duas características têm até agora não foram combinados. Aqui, descrevemos uma nova técnica para criar nano-pontos de anti-CD3 (um anticorpo que tem como alvo o complexo TCR) rodeado por uma bicamada lipídica com os lípidos que se difundem. A bicamada é depositada utilizando Langmuir-Blodgett / técnica de Langmuir-Schaefer 7, 15, 16 e, se desejado, pode ser funcionalizado com uma proteína específica - por exemplo, o ligando da integrina da célula T (chamado ICAM1). Além disso, os anti-CD3 pontos proteína could ser substituído com um outro anticorpo ou CAM. Enquanto nós escolhemos as proteínas para uso futuro como plataforma para estudos de adesão de células T, a estratégia detalhado aqui pode ser adaptado para qualquer proteína e mesmo DNA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass coverslips</td>
			<td>Assistent, Karl Hecht KG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Observation chamber</td>
			<td>Home made</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline surfactant concentrate (Hellmanex)</td>
			<td>Hella Analytics</td>
			<td>9-307-011-4-507</td>
		</tr>
		<tr>
			<td>Ultra-sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Labbox</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallizer&#160;</td>
			<td>Shott</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidine</td>
			<td>Thermo Fischer Scientifique</td>
			<td>84607</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Sigma-aldrich</td>
			<td>P3813</td>
		</tr>
		<tr>
			<td>Water MQ&#160;</td>
			<td>ELGA, Veolia France</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Corpuscular Inc</td>
			<td>147114-10</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma-aldrich</td>
			<td>A3648</td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma-aldrich</td>
			<td>A8549</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
		</tr>
		<tr>
			<td>Dansyl-PE</td>
			<td>Avanti Polar Lipids</td>
			<td>810330C</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-aldrich</td>
			<td>650471</td>
		</tr>
		<tr>
			<td>Gastight syringe&#160;</td>
			<td>Dominique Dustcher , France</td>
			<td>74453</td>
		</tr>
		<tr>
			<td>Film balance</td>
			<td>NIMA</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss, Germany</td>
			<td>TIRF-III system</td>
		</tr>
		<tr>
			<td>Aluminium Target&#160;</td>
			<td>Kurt J. Lesker Compagny, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Radio Frequency Magnetron sputtering Syst&#232;me&#160;</td>
			<td>modified SMC 600 tool by ALCATEL , France</td>
			<td></td>
		</tr>
	</tbody>
</table>

ligand nano-cluster arrays in a supported lipid bilayer

Actualmente há um grande interesse em criar matrizes ordenadas de ilhas de proteínas adesivas num mar do superficial passivada para estudos de biologia celular. Nos últimos anos, tornou-se cada vez mais claro que as células vivas responder, não só para a natureza bioquímica das moléculas que lhes são apresentados, mas também à maneira como essas moléculas são apresentados. Criando proteína micro-padrões é, portanto, agora um padrão em muitos laboratórios de biologia; nano-padrões também são mais acessíveis. No entanto, no contexto de interacções célula-célula, existe uma necessidade de padrão não só proteínas, mas também bicamadas lipídicas. Tal padronização dupla proteo-lipídica até agora não tem sido facilmente acessível. Oferecemos uma técnica fácil para criar proteínas nano-pontos suportados em vidro e propor um método para aterrar o espaço inter-ponto com uma bicamada lipídica suportado (SLB). A partir de foto-branqueamento de traçador lipídios fluorescentes incluídos no SLB, demonstramos que a bicamada exibe considerável-plfluidez ane. Funcionalização os pontos de proteínas com grupos fluorescentes nos permite imagem los e mostrar que eles são ordenados em uma rede hexagonal regular. O tamanho típico ponto é de cerca de 800 nm e o espaçamento aqui demonstrada é de 2 micra. Estes substratos são esperados para servir como plataformas úteis para a adesão celular, a migração e estudos mecano-sensores.

As imagens de fluorescência foram analisados ​​para medir o espaçamento e o tamanho dos pontos. Espaçamento típico verificou-se ser 1,900 ± 80 nm e dot-tamanho típico foi de 600 ± 100 nm (Figura 1 g). O espaçamento é definido pelo tamanho das contas utilizadas para a máscara. O ponto de tamanho é definido pelo, bem como condições de deposição do grânulo de tamanho. O SLB é depositada unicamente em torno dos pontos de proteína e não sobre elas <stro...

Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Apresenta-se um protocolo para funcionalizar vidro com manchas proteicas nanométricas rodeado por uma bicamada lipídica fluida. Estes substratos são compatíveis com microscopia óptica avançado e espera-se para servir como plataforma para estudos de adesão celular e de migração.

Matrizes ligando Nano-fragmentação em uma bicamada lipídica Compatível

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

ligand-nano-cluster-arrays-in-a-supported-lipid-bilayer

Research

JoVE Journal

Bioengineering

6.9K visualizações.  CNRS, UMR 7325, CINaM.  Apresenta-se um protocolo para funcionalizar vidro com manchas proteicas nanométricas rodeado por uma bicamada lipídica fluida. Estes substratos são compatíveis com microscopia óptica avançado e espera-se para servir como plataforma para estudos de adesão celular e de migração. 

Vídeo: Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Bioengenharia

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis ​​Líquido Análise Espectrometria de Massa-Cromatografia

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Método de extrusão constante pressão controlada para a Preparação de vesículas lipídicas nanométricas

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Investigando Sistemas receptor-ligante do Celulossoma com base-AFM única molécula Força Espectroscopia

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lipid Bilayer Vesicle Geração Usando Microfluidic jateamento

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Preparação de Mica Apoiado Lipid Bicamadas para alta resolução Microscopia Óptica de Imagem

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Projeção de imagem atômica e força Espectroscopia de Bicamadas Lipídicas suportados

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication pelo Solvent-assistida Lipid bicamada (SALB) Método

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.