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1. Mica e Slides Preparação <ol><li> Lugar No. 1 ½ (0,17 mm) em lamelas suporte de coloração. </li><li> Sonicar durante 30 min em 2% de detergente a 60 ° C. </li><li> Lave 20 vezes com água deionizada. </li><li> Retirar as lâminas usando uma pinça e secar com ar comprimido ou nitrogênio. </li><li> Corte folha mica em 10 x 10 mm pedaços quadrados usando uma tesoura ou lâmina de barbear. </li><li> Corte cada pedaço mica em 2-3 folhetos finas utilizando lâmina de barbear. NOTA: Esta etapa exige o uso de lâmina afiada. </li></ol> 2. Assembléia Mica e da Câmara de montagem <ol><li> Limpe lâmina de vidro microscópicas com etanol. </li><li> Glue folheto de mica cortado no passo 1.6 a lâmina de vidro com adesivo óptico. Adesivo de baixa viscosidade é aconselhável para melhor difundir a queda e cole a mica. </li><li> Cure sob a lâmpada UV, 10 min. NOTA: O adesivo é curado por luz UV com uma absorção máxima na gama de 350 a 380nm e a energia recomendado required para cura total é de 4,5 J / cm 2. No entanto, as diferentes fontes de luz pode ser utilizado para este passo (ver materiais fornecidos pelo fornecedor de adesivo). </li><li> Expor uma superfície mica limpo, eliminando as primeiras camadas com fita adesiva. </li><li> Coloque uma pequena gota (~ 20 mM) de adesivo óptico sobre a superfície da mica. NOTA: Nesta etapa, adesivo de alta viscosidade, com baixo nível de autofluorescência é aconselhada. Nossa experiência mostrou que o uso de combinação de baixo (passo 2.2) e adesivos de alta viscosidade aumenta significativamente a eficácia da divisão mica (passo 2.7). </li><li> Delicadamente, coloque lamela limpo de fresco para a gota de cola, evitando bolhas de ar, vamos contentar com 1 min. </li><li> Cure sob a lâmpada UV, 10 min. NOTA: após esta etapa, a sanduíche da lâmina de vidro, mica e lamela pode ser armazenado por um período relativamente longo de tempo (até algumas semanas). Vá para a próxima etapa, pouco antes da preparação real SLB, para certificar-se da superfície da mica é fresco. NOTA: O adesivo é curado por luz UV com uma absorção máxima na gama de 350 a 380nm e a energia necessária para recomendada a cura completa é 4.5J/cm 2. No entanto, as diferentes fontes de luz pode ser utilizado para este passo (ver materiais fornecidos pelo fornecedor de adesivo). </li><li> Usando um estilete, desmontar cuidadosamente a lamela de lâmina de vidro lateral, como mostrado no vídeo. Na maioria dos casos, uma camada fina e plana de mica permanecerá ligado à lamela. Mica ligado a lâmina de vidro podem ser reutilizados (repita a partir do passo 2.4). </li><li> Verificar a qualidade da superfície ao olho nu ou sob um microscópio de dissecação, a certificar-se de que a camada de mica ainda está colada à lamela e não foi completamente removido durante a cisão no passo 2.8. Fazendo um pequeno arranhão com uma pinça ou agulha de dissecação ajudará a distinguir entre o adesivo que tem uma consistência diferente detectável a partir de mica. </li><li> Remova a vedação de borracha a partir de um frasco de tampa de 1,5 ml e cola a tampa de cabeça para baixo para a superfície, usando ópticaadesiva ou unha polonês, e curar com lâmpada UV, ou deixar que o ar seco 10 min, respectivamente. NOTA: Se a amostra é preparada para simultânea óptico (TIRFM ou confocal) com imagens de AFM, um ml frasco cap 1.5 pode ser muito pequeno para montar a cabeça AFM no palco microscópio. Nesse caso, o tampão pode ser substituído por qualquer plástico junta tórica com um diâmetro maior, adequado para a montagem de cabeça de AFM. No caso, quando o experimento requer remoção da tampa, sem danificar a superfície de mica, graxa de silicone pode ser usado em vez de cola ou unha polonês. </li></ol> 3. Formação Apoiado Lipid Bilayer (SLB) <ol><li> Coloque solução de lipossomas preparados na hora na câmara com a superfície. O volume mínimo necessário para a formação de SLB é ~ 30 ul. NOTA: Para obter mais detalhes sobre o lipossoma e formação SLB, referem-se a protocolos publicados, tais como tais como Bolsa et al, 2014 15.. </li><li> Prossiga com formação SLB usando o protocolo desejado. Durante a incubação eimagiologia, a câmara pode ser colocada num bloco de calor ou fase de microscópio aquecida para manter a temperatura necessária para manter os lípidos sendo utilizadas acima da sua temperatura de fusão. </li></ol>

<ol><li>Giocondi, M. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+topography+of+membrane+domains%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta+%28BBA%29+-+Biomembranes%22%5BJournal%5D)">Surface topography of membrane domains.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 703-718 (2010).</li>
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<li>Bag, N., Sankaran, J., Paul, A., Kraut, R. S., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calibration+and+limits+of+camera-based+fluorescence+correlation+spectroscopy%3A+a+supported+lipid+bilayer+study%5BTitle%5D)+AND+%22Chemphyschem%3A+a+European+Journal+of+Chemical+Physics+and+Physical+Chemistry%22%5BJournal%5D)">Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study.</a> Chemphyschem: a European Journal of Chemical Physics and Physical Chemistry. 13, 2784-2794 (2012).</li>
<li>Singh, S., Keller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+force+microscopy+of+supported+planar+membrane+bilayers%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Atomic force microscopy of supported planar membrane bilayers.</a> Biophysical Journal. 60, 1401-1410 (1991).</li>
<li>Przybylo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid+Diffusion+in+Giant+Unilamellar+Vesicles+Is+More+than+2+Times+Faster+than+in+Supported+Phospholipid+Bilayers+under+Identical+Conditions%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Lipid Diffusion in Giant Unilamellar Vesicles Is More than 2 Times Faster than in Supported Phospholipid Bilayers under Identical Conditions.</a> Langmuir. 22, 9096-9099 (2006).</li>
<li>Scomparin, C., Lecuyer, S., Ferreira, M., Charitat, T., Tinland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusion+in+supported+lipid+bilayers%3A+influence+of+substrate+and+preparation+technique+on+the+internal+dynamics%5BTitle%5D)+AND+%22Eur+Phys+J+E+Soft+Matter%22%5BJournal%5D)">Diffusion in supported lipid bilayers: influence of substrate and preparation technique on the internal dynamics.</a> Eur Phys J E Soft Matter. 28, 211-220 (2009).</li>
<li>Oreopoulos, J., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+scanning+probe+and+total+internal+reflection+fluorescence+microscopy%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Combined scanning probe and total internal reflection fluorescence microscopy.</a> Methods. 46, 2-10 (2008).</li>
<li>Shaw, J. E., Slade, A., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+in+situ+total+internal+reflectance+fluorescence%2Fatomic+force+microscopy+studies+of+DPPC%2FdPOPC+microdomains+in+supported+planar+lipid+bilayers%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.</a> J Am Chem Soc. 125, 11838-11839 (2003).</li>
<li>Skaug, M. J., Faller, R., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correlating+anomalous+diffusion+with+lipid+bilayer+membrane+structure+using+single+molecule+tracking+and+atomic+force+microscopy%5BTitle%5D)+AND+%22J+Chem+Phys%22%5BJournal%5D)">Correlating anomalous diffusion with lipid bilayer membrane structure using single molecule tracking and atomic force microscopy.</a> J Chem Phys. 134, (2011).</li>
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<li>Linkert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metadata+matters%3A+access+to+image+data+in+the+real+world%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Metadata matters: access to image data in the real world.</a> J Cell Biol. 189, 777-782 (2010).</li>
<li>Schutz, G. J., Schindler, H., Schmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+microscopy+on+model+membranes+reveals+anomalous+diffusion%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Single-molecule microscopy on model membranes reveals anomalous diffusion.</a> Biophys J. 73, 1073-1080 (1997).</li>
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</ol>

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Este protocolo descreve um método para a preparação de superfícies lisas e de mica fina para deposição de bicamada lipídica e de alta resolução de imagem. A técnica requer habilidades manuais mínimos, limitado principalmente para a desmontagem cuidadosa do sanduíche vidro-mica-vidro (passo 2.8), que é fundamental para a obtenção de uma superfície de mica de alta qualidade. A inspecção da mica clivada de fresco é sempre necessário, neste ponto, uma vez que é possível para a mica de separar da subst?...

O objetivo geral do presente protocolo é mostrar um método para preparar superfícies de mica em alta resolução de imagem de mica suportada bicamadas lipídicas (EBSL) utilizando microscopia óptica de fluorescência total de reflexão interna (TIRFM) ou microscopia confocal, que também poderia ser combinada com a força atômica microscopia (AFM). EBSL é um modelo amplamente usado para numerosos estudos de agrupamento lipídico, a separação de fases, a dinâmica de componentes de bicamada ou as suas interacções com peptídeos, proteínas ou outros compostos 1-5. Substratos diferentes podem ser usadas para a formação de SLB (isto é, vidro, mica, dióxido de silício, polímeros), dependendo da natureza do estudo 4,6-8. Estudos de membrana típicas contam com técnicas de imagem com base em microscopia, como TIRFM e AFM. Assim, para a imagiologia TIRFM, uma superfície de vidro é uma escolha típica porque o vidro é transparente. Preparação de vidro é relativamente fácil, e a qualidade dos resultados é primariamentedeterminada pela superfície limpeza completa antes da deposição de vesículas lipídicas. AFM, devido à sua alta resolução axial requer superfícies mica. Mica é um mineral de silicato, com cerca de clivagem basal perfeita. Assim, a mica recém clivada é atomicamente plana, permitindo a observação da membrana diferenças de altura, mesmo em escala sub-nanométrica 9. Estudos de difusão, utilizando métodos como a espectroscopia de correlação de fluorescência (FCS), seguindo molécula única (SMT), e recuperação de fluorescência após a fotodegradação (FRAP) mostrou no entanto, que a dinâmica da membrana lipídica depender muito do tipo de superfície sobre a qual são depositados, em que vidro mica e pode dar resultados muito diferentes 10,11. Estas diferenças incluem não apenas os coeficientes de difusão das sondas de membrana, mas também a detecção de populações distintas de partículas que difundem a taxas diferentes, e, possivelmente, a comutação entre os diferentes estados. Assim,a comparação directa dos resultados obtidos utilizando técnicas de TIRFM e AFM é muitas vezes problemática, a menos que a mesma superfície (neste caso, a mica) é usado. Embora existam alguns estudos onde TIRFM e AFM bicamada de imagem foi realizado na mesma superfície mica 12,13, mica é raramente usado para a microscopia óptica, principalmente por causa de problemas de manuseio. Preparação mica requer corte à mão em folhetos finos, que são então coladas à lamela utilizando adesiva óptica 12. Este método, porém, requer um pouco de prática para alcançar resultados satisfatórios. Além disso, as superfícies obtidas são muitas vezes ondulado e grosso, tornando-os difíceis de usar com baixa distância de trabalho, lentes de alta abertura numérica. Mica superfícies preparadas como descrito no presente protocolo são muito finas (~ 220 mM, incluindo a espessura da lamela de 170 um) e extremamente lisa, evitando &quot;ondulação&quot;, o qual é crítico para uma imagem com alta resolução. Eles podem ser utilizadospara TIRFM ou confocal setups. Além disso, as mesmas amostras podem ser transferidos para a AFM, e ainda visualizados simultaneamente com TIRFM / confocal e AFM. A combinação destas duas técnicas permite correlação direta do comportamento de difusão com a estrutura da membrana de bicamada 14. Como as superfícies de mica são recém-clivada, são limpos e não necessitam de demorado, pouco reprodutível, e procedimentos de limpeza potencialmente perigosas (protocolos de limpeza de vidro geralmente incluem produtos químicos como solução Piranha, ácido sulfúrico, hidróxido de sódio / potássio). Montagem de uma pequena câmara, também descrito no protocolo, reduz o volume de vesículas necessários para a formação de bicamada eficaz para menos de 50 ul. Finalmente, todo o processo de montagem de superfície não é demorado (preparação leva menos do que 30 min), e não requerem um elevado grau de habilidade manual, tal como a clivagem de mica convencional e colagem.

<table><tbody><tr><td>Bath Sonicator</td><td>Fisher Scientific</td><td>FB15051</td><td></td></tr><tr><td>Coverslips 24 x 50 mm - No H1.5</td><td>Marienfeld</td><td>102222</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850357</td><td></td></tr><tr><td>Hellmanex III (detergent)</td><td>Hellma Analytics</td><td>320.003</td><td></td></tr><tr><td>Mica V-1 Grade</td><td>SPI Suppliers</td><td>1872-CA</td><td></td></tr><tr><td>Optical Adhesive (high viscosity)</td><td>Norland Products</td><td>NOA63</td><td></td></tr><tr><td>Optical Adhesive (low viscosity)</td><td>Norland Products</td><td>NOA60</td><td></td></tr><tr><td>Sphingomyelin-ATTO647N</td><td>AttoTec</td><td>AD 647N-171</td><td></td></tr><tr><td>UV lamp</td><td>Synoptics Ltd.</td><td>GelVue GVM20</td><td>The lamp was set to 100% power</td></tr></tbody></table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

Bicamadas lipídicas suportadas (EBSL) são amplamente utilizados como modelo para o estudo de propriedades da membrana (separação de fases, de agrupamento, dinâmica) e sua interação com outros compostos, tais como drogas ou peptídeos. No entanto características SLB diferente, dependendo do suporte utilizado. Técnicas comumente usados ​​para imagens e medições SLB são microscopia única molécula de fluorescência, FCS e microscopia de força atômica (AFM). Como a maioria dos estudos de imagiologia óptica são realizadas sobre um suporte de vidro, enquanto que a AFM requer uma superfície extremamente lisa (geralmente mica), os resultados destas técnicas não podem ser directamente comparados, uma vez que as propriedades de carga e de suavidade destes materiais influenciam fortemente a difusão. Infelizmente, o nível elevado de destreza manual necessária para o corte e colagem de fatias finas de mica para a lâmina de vidro apresenta um obstáculo para a utilização de rotina de mica para a preparação SLB. Embora este seja o método de escolha, tal mica preparadasuperfícies muitas vezes acabam por ser irregular (ondulado) e difícil de imagem, especialmente com distância de trabalho pequeno, lentes de alta abertura numérica. Aqui apresentamos um método simples e reprodutível para preparar superfícies planas finas de mica para a deposição de vesícula lipídica e preparação SLB. Além disso, a nossa câmara personalizado feito requer apenas pequenas quantidades de vesículas para a formação SLB. Os resultados do procedimento geral para a produção eficiente, simples e barata de alta qualidade superfícies da bicamada lipídica, que são directamente comparáveis ​​com os utilizados em estudos de AFM.

O comportamento de difusão de sondas fluorescentes lipídicas em EBSL é diferente, dependendo do substrato. TIRFM combinada com a técnica SMT é um método valioso para a visualização de movimentos de partículas e extrair os seus coeficientes de difusão. Sinais de moléculas isoladas de uma sonda de esfingomielina-ATTO647N difusão numa DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) em bicamada suportada em vidro e de mica são mostrados na figura animada em anexo. A superfície de mica foi preparado de ac...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

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

Apresenta-se um método de preparação de mica suportado bicamadas lipídicas para microscopia de alta resolução. Mica é transparente e liso em uma escala atômica, mas raramente usado em imagiologia por causa de dificuldades de manuseio; a preparação resulta em deposição uniforme da folha de mica, e reduz o material usado na preparação de bicamada.

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 ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

13.4K visualizações.  Nanyang Technological University.  Apresenta-se um método de preparação de mica suportado bicamadas lipídicas para microscopia de alta resolução. Mica é transparente e liso em uma escala atômica, mas raramente usado em imagiologia por causa de dificuldades de manuseio; a preparação resulta em deposição uniforme da folha de mica, e reduz o material usado na preparação de bicamada. 

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

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Suportados Bicamadas Planar para a Formação do Estudo da Sinapses imunológicas e Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Biologia

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Formação de biomembrana Microarrays com um método de montagem baseada em Rodo

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengenharia

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 ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-Resolution Imaging da Célula Natural Killer imunológica Synapse em um vidro com suporte Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Imunologia e Infecção

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Difusão e Montagem de Molécula Única em Membranas Lipídicas Repletas de Polímeros

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Bioquímica

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

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule 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 ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

Reconstituição de Córtices de Actina Mínima Amarrados à Membrana em Bicamadas Lipídicas Suportadas

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

Ensaio de Bicamada Lipídica Planar: Uma Técnica In Vitro Baseada em Microscopia para Investigar a Montagem de Septina em Mimetizadores de Membrana Biológica

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...