Name: ligand nano-cluster arrays in a supported lipid bilayer
Uploaded: 2017-04-23T14:00:00
Description: Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

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 ?...

<ol>
	<li>Alberts, B., Johnson, A., Lewis, 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=.">.</a> Molecular Biology of the Cell. , (2002).</li><li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cell+Receptor-Proximal+Signals+Are+Sustained+in+Peripheral+Microclusters+and+Terminated+in+the+Central+Supramolecular+Activation+Cluster.">T Cell Receptor-Proximal Signals Are Sustained in Peripheral Microclusters and Terminated in the Central Supramolecular Activation Cluster.</a> Immunity. 25 (1), 117-127 (2006).</li><li>Kaizuka, Y., 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=Mechanisms+for+segregating+T+cell+receptor+and+adhesion+molecules+during+immunological+synapse+formation+in+Jurkat+T+cells.">Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells.</a> Proc Natl Acad Sci USA. 104 (51), 20296-20301 (2007).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li><li>Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell-antigen+recognition+and+the+immunological+synapse.">T-cell-antigen recognition and the immunological synapse.</a> Nat Rev Immunol. 3 (12), 973-983 (2003).</li><li>Grakoui, A., 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=The+Immunological+Synapse:+A+Molecular+Machine+Controlling+T+Cell+Activation.">The Immunological Synapse: A Molecular Machine Controlling T Cell Activation.</a> Science. 285, 221-228 (1999).</li><li>Dillard, P., Varma, R., Sengupta, K., Limozin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-mediated+friction+determines+morphodynamics+of+spreading+T+cells.">Ligand-mediated friction determines morphodynamics of spreading T cells.</a> Biophys J. 107 (11), 2629-2638 (2014).</li><li>Lu, X., 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=Endogenous+viral+antigen+processing+generates+peptide-specific+MHC+class+I+cell-surface+clusters.">Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.</a> Proc Natl Acad Sci U S A. 109 (38), 15407-15412 (2012).</li><li>Pi, F., Dillard, P., 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=Size-Tunable+Organic+Nanodot+Arrays:+A+Versatile+Platform+for+Manipulating+and+Imaging+Cells.">Size-Tunable Organic Nanodot Arrays: A Versatile Platform for Manipulating and Imaging Cells.</a> Nano Lett. 15 (8), 5178-5184 (2015).</li><li>Deeg, 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=T+cell+activation+is+determined+by+the+number+of+presented+antigens.">T cell activation is determined by the number of presented antigens.</a> Nano Lett. 13 (11), 5619-5626 (2013).</li><li>Delcassian, D., 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=Nanoscale+ligand+spacing+influences+receptor+triggering+in+T+cells+and+NK+cells.">Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.</a> Nano Lett. 13 (11), 5608-5614 (2013).</li><li>Matic, J., Deeg, J., Scheffold, A., Goldstein, I., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+tuning+and+efficient+T+cell+activation+with+stimulatory+aCD3+nanoarrays.">Fine tuning and efficient T cell activation with stimulatory aCD3 nanoarrays.</a> Nano Lett. 13 (11), 5090-5097 (2013).</li><li>Dillard, P., Pi, F., Lellouch, A. C., Limozin, L., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-clustering+of+ligands+on+surrogate+antigen+presenting+cells+modulates+T+cell+membrane+adhesion+and+organization.">Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.</a> Integr Biol. 8 (3), 287-301 (2016).</li><li>Pi, F., Dillard, P., Limozin, L., Charrier, A., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometric+protein-patch+arrays+on+glass+and+polydimethylsiloxane+for+cell+adhesion+studies.">Nanometric protein-patch arrays on glass and polydimethylsiloxane for cell adhesion studies.</a> Nano lett. 13 (7), 3372-3378 (2013).</li><li>Fenz, S. F., Merkel, R., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+intermembrane+distance:+case+study+of+avidin+and+E-cadherin+mediated+adhesion.">Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.</a> Langmuir. 25 (2), 1074-1085 (2009).</li><li>Sengupta, K., 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=Mimicking+tissue+surfaces+by+supported+membrane+coupled+ultra-thin+layer+of+hyaluronic+acid.">Mimicking tissue surfaces by supported membrane coupled ultra-thin layer of hyaluronic acid.</a> Langmuir. 19 (5), 1775-1781 (2003).</li><li>Taylor, Z. R., Keay, J. C., Sanchez, E. S., Johnson, M. B., Schmidtke, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independently+controlling+protein+dot+size+and+spacing+in+particle+lithography.">Independently controlling protein dot size and spacing in particle lithography.</a> Langmuir. 28 (25), 9656-9663 (2012).</li><li>Massou, S., 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=Large+scale+ordered+topographical+and+chemical+nano-features+from+anodic+alumina+templates.">Large scale ordered topographical and chemical nano-features from anodic alumina templates.</a> Appl. Surf Sci. 256 (2), 395-398 (2009).</li><li>Selhuber-Unkel, C., Lopez-Garcia, M., Kessler, H., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperativity+in+adhesion+cluster+formation+during+initial+cell+adhesion.">Cooperativity in adhesion cluster formation during initial cell adhesion.</a> Biophys J. 95 (11), 5424-5431 (2008).</li><li>Arnold, M., 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=Induction+of+cell+polarization+and+migration+by+a+gradient+of+nanoscale+variations+in+adhesive+ligand+spacing.">Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing.</a> Nano Lett. 8 (7), 2063-2069 (2008).</li><li>Cavalcanti-Adam, E. A., 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=Cell+spreading+and+focal+adhesion+dynamics+are+regulated+by+spacing+of+integrin+ligands.">Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.</a> Biophys J. 92 (8), 2964-2974 (2007).</li><li>Schvartzman, M., 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=Nanolithographic+Control+of+the+Spatial+Organization+of+Cellular+Adhesion+Receptors+at+the+Single-Molecule+Level.">Nanolithographic Control of the Spatial Organization of Cellular Adhesion Receptors at the Single-Molecule Level.</a> Nano Lett. 11 (3), 1306-1312 (2011).</li><li>Mossman, K., Groves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+supported+membranes+as+tools+for+quantitative+studies+of+the+immunological+synapse.">Micropatterned supported membranes as tools for quantitative studies of the immunological synapse.</a> Chem.Soc.Rev. 36 (1), 46-54 (2007).</li><li>Furlan, G., 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=Phosphatase+CD45+both+positively+and+negatively+regulates+T+cell+receptor+phosphorylation+in+reconstituted+membrane+protein+clusters.">Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.</a> J Biol Chem. 289 (41), 28514-28525 (2014).</li><li>Hsu, C. 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 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 ...

The overall goal of this technique is to fabricate an array of protein nano-clusters in a supported lipid bilayer, which is compatible with sensitive surface microscopy techniques for cell biology applications. This method can help answer key questions in the field of cell biophysics, in particular, how the nano-clustering of ligands influences T cell activation and adhesion. The main advantage of this technique is that it is easily reproducible in standard biophysical lab and it is compatible with advanced surface sensitive microscopy technique, such as TIRF and RICM.Though this technique is designed to provide insight into immunology, it can be applied to other cell types with potential applications in cell biology, oncology and tissue engineering. To begin this procedure, clean the glass cover slides and observation chambers as outlined in the text protocol. Then, deposit 70 microliters of 2%silica bead suspension drop by drop onto a cover slide held at a 15 degree incline.Flip the glass 90 degrees every 15 seconds for one minute while the suspensions dries. It is critical to create a closely-packed monolayer of beads and to avoid the formation of multilayers and oil clusters. That's why the concentration and the volume of the bead solution as well as the hydrophilicity of the slide need to be optimized.After the liquid has evaporated, place the slide inside a radio frequency magnatron sputtering device onto a rotating table situated 105 millimeters away from an aluminum silicon target. After this, use a turbo-molecular pump to decrease the pressure in the deposition chamber to 2.6 times 10 to the negative fourth pascals. Introduce a pure argon atmosphere with a flux of 10 standard cubic centimeters per minute and a pressure of 0.8 pascals.Next, switch on the radio frequency power generator. After the plasma is stabilized, sputter for two minutes while keeping the shutter closed, to remove possible impurities from the target surface. Open the shutter, and allow the sputtering to continue for 60 minutes to deposit a 200-nanometer thick layer of aluminum on the slide.Then cut the flow of argon. Close the gate valve to isolate the turbo-molecular pump from the deposition chamber. After this, vent the chamber with clean nitrogen to reach ambient pressure.Recover the aluminum-coated slide from the chamber. Immerse the recovered slide in ultra-pure water at room temperature and ultra-sonicate at 50 watts and 50 hertz for 30 seconds. Next, deposit 0.5 milliters of APTES at the bottom of a desiccator.Place the glass slide on a ceramic grid. Place the grid into the desiccator. Connect the desiccator to a membrane pump.Then run the pump at maximum power for 30 minutes to generate a low vacuum. Close the valve of the desiccator, and switch off the pump. Heat to 50 degrees celsius for one hour.After this open the desiccator and collect the slide. Place the collected slide on a PTFE support. Deposit 2 milliliters of 25 micrograms per milliliter biotin dissolved in PBS.Allow the slide to rest for 30 minutes at room temperature. Then rinse 10 times with PBS. Incubate the slide in a solution of sodium hydroxide and PBS at room temperature overnight.After this, rinse the slide 10 times with ultra-pure water. After cleaning the Langmuir trough, place PTFE trays in the trough's PTFE enclosure. Then fill it with ultra-pure water.Set the measured pressure to zero millinewtons per meter. Using a gas-tight glass metal syringe, deposit 30 microliters of 1 milligram per millimeter DOPC and chloroform solution on the water's surface. Close the PTFE barrier until the desired pressure of 27 millinewtons per meter is reached to compress the lipid monolayer.Next, using a motorized clamp, dip the prepared glass slide into the PTFE enclosure. Hold the slide in the clamp perpendicular to the air-water interface. Raise the slide through the interface at a rate of 15 millimeters per minute while maintaining a constant pressure of 27 millinewtons per meter.Then, place the glass slide horizontally on the surface of the water above a PTFE tray. Next, using metal tweezers, push the slide down into the PTFE tray, immersing it in the ultra-pure water. Use the tweezers to transfer the PTFE tray containing the slide into a crystallizer filled with ultra-pure water.Transfer the coated slide underwater into an observation chamber. After this, close the chamber, ensuring that approximately 1 milliliter of water is trapped inside. Another delicate step is the assembly of the sample chamber underwater.And one should absolutely avoid contact with the air of the deposited bilayers, so to ensure this use a large volume of water and ensure that the whole assembly process can occur underwater. Remove the assembled chamber from the water, check to ensure that the chamber is watertight and free from leaks. Add 500 microliters of PBS, then remove 500 microliters of liquid from the chamber.Repeat this process 10 times to fully replace the one milliliter of ultra-pure water in the chamber with PBS. Next, introduce 100 microgram per milliliter of bovine serum albumen to the observation chamber. Incubate at room temperature for 30 minutes.After this, rinse the bilayer by removing and adding 500 microliters from the chamber, 10 times. Then, functionalize with ligands, deposit cells, and observe the proteolipidic nanopattern and SLB fluidity as outlined in the text protocol. In this protocol, a bead mask is used to create a secondary metal mask by controlled deposition of aluminum.Then, the bead mask is removed, leaving a layer of aluminum with holes. Next, an organo-amino siline, called APTES, is deposited in the vapor phase, followed by deposition of BSA-Biotin in aqueous phase. Then, aluminum is removed, revealing nanometric protein patches.Finally the inter-dot space is back-filled with a supported lipid bilayer. Epiflourescence images are then taken of the nano-dots and supported lipid bilayer. A composite image shows perfect complementarity with the lipid bilayer deposited uniquely around the protein dots, but not on them.In an epiflourescence image of a freshly-deposited supported lipid bilayer, the protein dots appear as dark spots in a bright sea of lipids. However, after continuous photo bleaching for 50 seconds, the image shows a halo inside the region delimited by the field diaphragm, indicating that the lipids are mobile. Analysis of the average intensity profile along the edge of the field diaphragm, and the decay of this intensity over time during the bleaching process, reveals that the diffusion constant is five micrometers squared per second.This technique can be done in two days if it is performed properly. Its's important to maintain very clean conditions during aluminum deposition, and to carefully calibrate the deposition curve. Following this procedure, the patterned slide can be further functionalized.For example, by adding another biomolecule in the bilayer. We use this to mimic the antigen plant-eating cells, in order to study the T cell activation. So after its development, this technique should interest diverse audiences.For example, tissue engineers may want to use it as scaffold to grow artificial tissues, and oncologists may use this as a platform for posing fundamental questions about addition of cancer cells. After watching this video you should have a good understanding of how to fabricate an area of protein nano-clusters in supported lipid bilayer, which is compatible with an advanced microscopy technique. While we use this to study T cells, we believe that this substrate have the potential to become the platform of choice to study the interaction of any kind of cells with controlled pattern proteolipidic membrane.

Research

JoVE Journal

Bioengineering

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

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 ...

Bioengenharia

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

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

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

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

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

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

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

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 ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Formação Automated Lipid bicamada da membrana Usando um polidimetilsiloxano Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

Montagem controlada por temperatura e caracterização de um Bilayer de interface de gotícula

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Montagem de Modelos de Bicamadas lipídicas apoiadas e suspensas para o estudo de interações moleculares

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...