Este trabalho foi apoiado pelo Programa de Descoberta do Conselho de Pesquisa Australiano (ARC) (DP150101065) e pelo Arc Research Hub for Integrated Device for End-user Analysis at Low-levels (IDEAL) (IH150100028).

1. preparação de eletrodos tBLMs
<ol><li>Preparação do primeiro revestimento de monocamadas<ol>
<li>Mergulhe um slide de microscópio eletrodo padrão de ouro recém-sputtered em uma solução etanolica composta por uma razão de 3 mM 1:9 de benzil-dissulfeto-tetra-etilenoglicol-OH "espaçador" (dissulfeto benzilo composto por um spacer de quatro oxigênio-etileno, terminou com um grupo OH) e benzil-dissulfeto (tetra-etilenoglicol) n=2 C20-fitanyl moléculas "amarradas". Isso cria o primeiro revestimento de camada para o qual um bicamado pode ser ancorado. NOTA: O eletrodo de ouro é feito evaporando 100 nm, 99,9995% de ouro (5n5 ouro) em lâminas personalizadas de 25 mm x 75 mm de policarbonato20.</li>
		<li>Incubar eletrodos com a primeira camada à temperatura ambiente por pelo menos 1 h.</li>
		<li>Enxágüe os eletrodos de ouro imergindo em quantidades abundantes de etanol puro acima de 30 s.</li>
		<li>Use o slide de eletrodo dourado com a primeira monocamada diretamente para o próximo passo ou armazene em um pote cheio de etanol puro.</li>
		<li>NOTA: Para garantir a integridade da primeira camada, minimize qualquer contato direto com as porções de ouro do slide</li>
	</ol></li>
	<li>Montando o primeiro slide revestido de monocamada<ol>
<li>Retire cuidadosamente um slide de eletrodo de ouro coplanar de seu recipiente usando pinças, tendo certeza de não fazer contato com as áreas padronizadas onde os tBLMs se formarão. NOTA: Esteja atento para identificar o lado do slide sobre o qual o ouro é depositado.</li>
		<li>Lâmina seca de ar por 1 - 2 min para remover qualquer etanol residual.</li>
		<li>Coloque o eletrodo dourado sobre uma superfície seca, certifique-se de que o eletrodo dourado seja corretamente orientado com superfície dourada estampada voltada para cima.</li>
		<li>Descasque a tampa da camada adesiva transparente de um laminado fino e coloque sobre os 6 canais para definir cada poço.</li>
		<li>Use um rolo de pressão para liberar qualquer ar entre o slide e a camada adesiva transparente, conforme mostrado na Figura 1A. NOTA: O tempo necessário para esta etapa precisará ser otimizado pelo pesquisador. Neste protocolo, os tempos variam de 2 a 3 minutos.</li>
		<li>Introduza assim que possível (dentro de 1-2 minutos) a segunda bicamada lipídica ao primeiro eletrodo revestido de monocamada montado para auto-montagem para evitar danificar a primeira camada.</li>
	</ol></li>
	<li>Preparação do segundo bicamógrafo lipídudo<ol>
<li>Adicione 6 μL de lipídios de 3 mM de interesse ao primeiro poço do slide de seis poços. Não deixe que a borda da ponta da micropipette toque na superfície dourada, o que pode danificar as químicas amarradas no eletrodo. NOTA: A mistura lipídica utilizada neste trabalho consistiu em 3 mM 70% zwitterionic C20 diphytanyl-ether-glycero-fosphasphatidylcholina (DPEPC) e 30% Lipídios de diphytanyldiglyceride C20 (GDPE) misturados com 3 mM de colesterol-PEG-Biotina na razão molar 50:1.</li>
		<li>Introduza 6 μL da mistura lipídica aos outros poços com uma diferença de 10 s entre cada adição.</li>
		<li>Incubar cada poço por exatamente 2 minutos à temperatura ambiente antes de trocar a mistura lipídica sobre os eletrodos com um tampão como PBS. Estoque os tempos para a adição e troca de buffer 10 s separados para que cada poço seja incubado com o lipídio por exatamente 2 min cada.</li>
		<li>Lave mais 3 vezes com 50 μL de tampão PBS (pH 7.0). Certifique-se de deixar 50 μL de tampão sobre os eletrodos o tempo todo. Não deixe os eletrodos secarem. NOTA: Deslocar o solvente de etanol com a solução aquosa desta forma (o método de troca de solventes) permite a rápida formação de um único bicamado lipídeca ancorado no eletrodo de ouro através das químicas amarradas.</li>
	</ol></li>
	<li>Testando a formação do TBLM usando medições de espectroscopia de impedância elétrica (EIS)<ol>
<li>Insira slide de eletrodo preparado em um espectrômetro de impedância CA (por exemplo, Tethapod). Certifique-se de que o espectrômetro esteja conectado através de uma porta USB a um computador que executa o software.</li>
		<li>Abra o software, clique em Configurar e abra hardware.</li>
		<li>Defina as configurações de hardware para usar a excitação AC de pico ao pico de 25 mV.</li>
		<li>Definir frequências entre 0,1 e 10.000 Hz com dois passos por década para medidas de impedância rápida pressione ok.</li>
		<li>Clique no menu Configuração e abra o Modelo.</li>
		<li>Use um modelo de circuito equivalente que descreve o eletrodo de ouro amarrado como um elemento de fase constante em série com um resistor descrevendo o tampão eletrólito e uma rede de resistor-capacitor paralelo para descrever a bicamada lipídica, e pressione OK.</li>
		<li>Pressione o botão Iniciar para iniciar uma medição em tempo real da capacitância da membrana (Cm) e da condução da membrana (Gm). Os valores Cm dos tBLMs típicos devem estar na faixa de 12,5 nF a 15,5 nF para 10% de químicas amarradas21,22.</li>
		<li>Depois de executar o protocolo e terminar o experimento, salve os dados.</li>
		<li>Repita a medição com o próximo poço.</li>
	</ol></li>
</ol>2. Irradiação a laser
<ol><li>Configuração experimental NOTA: O sistema personalizado é configurado para cada tBLM bem individualmente.<ol>
<li>Realize experimentos em uma caixa à prova de luz para minimizar o laser perigosamente.</li>
		<li>Use uma tabela óptica para configurar o experimento para reduzir vibrações indesejadas.</li>
		<li>Coloque o leitor de impedância, onde está o slide de ouro conectado, em um estágio XYZ e eleve de tal forma que ele se sente no caminho da fonte laser.</li>
		<li>Use engrenagens microscópicas de foco grosseiras para controlar a altura da fonte laser para obter a precisão adequada.</li>
		<li>Direja o caminho laser ao longo do eixo longitudinal do slide do eletrodo. ATENÇÃO: Use sempre óculos de segurança a laser adequados e mantenha bons protocolos de segurança a laser.</li>
		<li>Permita que o laser ajustado selecionado se estabilize antes de iniciar o experimento. NOTA: Um esquema da configuração experimental é ilustrado na Figura 2A.</li>
	</ol>
</li>
	<li>Alinhamento de eletrodos a laser e ouro NOTA: Antes de começar, avalie sempre a saída de energia do laser usando um medidor de potência para garantir que apenas potências muito baixas sejam entregues aos tBLMs.<ol>
<li>Ajuste o caminho do laser ou o ângulo do eletrodo de modo que o laser passe pelo líquido que cobre o eletrodo e seja apenas visível, uniformemente, na superfície dourada.</li>
		<li>Ajuste a posição de luz do feixe de laser para cada experimento, levantando ou baixando a fonte do feixe de laser usando o ajuste fino enquanto observa alterações na condução da membrana.</li>
		<li>Bloqueie o botão para fixar a posição do caminho do laser quando não forem observadas alterações de condução. NOTA: O aumento dos valores de condutância da membrana será gerado quando o laser interagir com o eletrodo de ouro subjacente. É, portanto, importante ajustar o caminho do laser para que tais interações não sejam possíveis.</li>
	</ol>
</li>
	<li>Preparação da amostra<ol>
<li>Prepare o alinhamento da luz do feixe de laser (onde não há alteração na conduance da membrana), como mostrado na Figura 2, posição 3.</li>
		<li>Adicione GNPs de interesse (funcionalizados ou nus) ao buffer PBS no qual os tBLMs estão imersos enquanto o laser é desligado.</li>
		<li>Misture o buffer PBS ao redor dos tBLMs suavemente três vezes, tomando cuidado para não tocar no eletrodo.</li>
		<li>Incubar por 5-10 min em temperatura ambiente.</li>
		<li>Ligue o laser para irradiar a amostra, usando a posição correta de luz do feixe de laser alinhada, como visto na Figura 2, posição 3.</li>
		<li>Use a combinação apropriada de tamanho, forma e concentração de GNPs com comprimento de onda de luz laser. NOTA: O feixe laser do comprimento de onda definido deve acoplado à frequência de ressonância plasmon GNP correspondente.</li>
		<li>Registro medido continuamente (medições em tempo real).</li>
		<li>Realizar as etapas 2.2.1 - 2.3.7, omitindo a adição de GNP para os experimentos de controle.</li>
	</ol>
</li>
</ol>3. Análise e apresentação de dados estatísticos
<ol><li>Exporte os dados para uma planilha.</li>
	<li>Extrair o parâmetro de condutância da membrana versus o tempo.</li>
	<li>Use os dados gravados após definir uma luz de feixe de laser com a posição certa e antes da introdução dos GNPs.</li>
	<li>Normalize os dados dividindo a conduciência da membrana medida sobre a conduciência da membrana de base. NOTA: Isso confirma que alterações relativas nos valores de condução da membrana provocadas por GNPs irradiados introduzidos.</li>
	<li>Apresentar dados como parcelas de tempo (x-eixo) versus condução de membrana normalizada (eixo y).</li>
</ol>4. Prever a quantidade de calor localizado gerado nos tBLMs a partir de nanopartículas irradiadas (modelo preditivo térmico)
<ol><li>Resolva o problema da transferência de radiação de acordo com Dombrovsky23, a fim de calcular o poder de radiação absorvido em soluções de nanopartículas irradiadas.</li>
	<li>Calcule a geração de calor incorporando a fonte de calor devido à radiação absorvida na equação de energia. NOTA: Para uma explicação detalhada da análise numérica da geração de calor nos tBLMs a partir de nanopartículas irradiadas e da interface nanomaterial-eletrodo, consulte 19.</li>
</ol>

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Os autores declaram os seguintes interesses financeiros/relações pessoais, que podem ser considerados como potenciais interesses concorrentes: Prof. Bruce Cornell é Diretor - Ciência e Tecnologia na Surgical Diagnostics SDx tethered membranas Pty. Ltd.

Este protocolo descreve o uso do modelo tBLM com um substrato de eletrodos coplanar em conjunto com uma configuração de alinhamento a laser horizontal que permite a gravação de impedância elétrica em tempo real em resposta à irradiação a laser de nanopartículas de ouro. O método de gravação do EIS apresentado aqui constrói uma lista mínima de experimentos necessários para fornecer o registro de mudanças de corrente de íon em toda a membrana, o que corresponde ao calor ge...

O desempenho hipertérmico de nanomateriais de ouro irradiado oferece uma nova classe de tratamento minimamente invasivo, seletivo e direcionado para infecções e tumores1. O emprego de nanopartículas que podem ser aquecidas por um laser tem sido usado para destruir seletivamente células doentes, bem como fornecer um meio para o parto seletivo de medicamentos2,3. Uma consequência dos fenômenos da fototermólise de nanopartículas plasmônicas aquecidas é danos às membranas celulares. A membrana de bicamadas lipídicas fluidas é considerada um local particularmente vulnerável para as células submetidas a tais tratamentos, pois a desnaturação de proteínas de membrana intrínseca, bem como danos à membrana, também pode levar à morte celular4, já que muitas proteínas estão lá para manter o gradiente potencial iônico entre as membranas celulares. Embora a capacidade de determinar e monitorar a transferência de calor na nanoescala seja de interesse fundamental para o estudo e aplicação de GNPs irradiados1,5,6,7, avaliação e compreensão das interações moleculares entre GNPs e bio-membranas, bem como as consequências diretas dos fenômenos de aquecimento induzidos a laser de GNPs incorporados em tecidos biológicos, ainda estão para ser totalmente elucidado8. Portanto, uma compreensão completa do processo de hipertermia dos GNPs irradiados continua a ser um desafio. Como tal, o desenvolvimento de uma interface nanomaterial-eletrodo que imita o ambiente natural das células poderia fornecer um meio para realizar uma investigação aprofundada das características de transferência de calor de nanopartículas de ouro irradiadas dentro de sistemas biológicos.
A complexidade das membranas celulares nativas é um dos desafios significativos na compreensão das interações irradiadas de GNPs nas células. Existem várias plataformas de membrana artificial desenvolvidas para fornecer versões bio miméticas simples de arquitetura e funcionalidade de membrana lipídica natural, incluindo, mas não se limitando a, membranas lipídicas pretas9,membranas bicamadas apoiadas10,membranas bicamadas híbridas11,membranas lipídicas com almofadas de polímero12 e membranas lipídicas bicamadasamarradas 13. Cada modelo de membrana lipídica artificial tem vantagens e limitações distintas no que diz respeito à imitação das membranas lipídicas naturais14.
Este estudo descreve o emprego de eletrodos revestidos de membrana lipídica como um sensor para avaliar as interações de nanopartículas de ouro e membrana lipídica, utilizando o modelo tBLM. O esquema de detecção de biosensor baseado em tBLM fornece estabilidade e sensibilidade inerentes13, pois as membranas amarradas podem se auto-reparar, ao contrário de outros sistemas (como membranas formadas por patch-clamp ou lipossomos) em que apenas uma pequena quantidade de dano de membrana resulta em seu colapso15,16,17,18. Além disso, como os tBLMs são de dimensões mm2, a impedância de fundo é ordens de magnitude inferiores às técnicas de gravação de grampos, o que permite o registro de alterações no fluxo iônico da membrana basal devido às interações com nanopartículas. Como resultado disso, o presente protocolo pode contrastar mudanças na condutância da membrana por GNPs vinculados que são animados por lasers cujos poderes são tão baixos quanto 135 nW/μm2.
O sistema aqui apresentado fornece um método sensível e reprodutível para determinar parâmetros precisos de laser, tamanho de partículas, revestimentos de partículas e composição necessários para projetar e desenvolver terapias térmicas. Isso é fundamental para o refinamento de terapias fototérmicas emergentes, além de oferecer informações valiosas para mecanismos detalhados de transferência de calor dentro de sistemas biológicos. O protocolo apresentado baseia-se no trabalho publicado anteriormente19. Um esboço do protocolo é o seguinte: a primeira seção define a formação tBLM; a segunda seção descreve como construir a configuração e alinhar a fonte de laser de excitação; a seção final ilustra como extrair informações dos dados de espectroscopia de impedância elétrica.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 nm diameter streptavidin-conjugated gold nanoparticles</td>
			<td>Cytodiagnostics</td>
			<td>AC-30-04-05</td>
			<td>This is a streptavidin-conjugated GNPs product ready for use</td>
		</tr>
		<tr>
			<td>30 nm diameter bare gold nanoparticles</td>
			<td>Sigma-Aldrich</td>
			<td>753629</td>
			<td>This is a bare GNPs product ready for use</td>
		</tr>
		<tr>
			<td>Cholesterol-PEG-Biotin (MW1000)</td>
			<td>NANOCS</td>
			<td>PG2-BNCS-10k</td>
			<td>Dissolved in highly pure ethanol</td>
		</tr>
		<tr>
			<td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S1</td>
			<td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Spacer molecules</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&#160;</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Tethered molecules</td>
		</tr>
		<tr>
			<td>532 nm green laser continuous light</td>
			<td>OBIS LS/OBIS CORE LS, China</td>
			<td>ND-1000</td>
			<td>The power of this laser was ~135&#160;mW&#160;</td>
		</tr>
		<tr>
			<td>tethaPod EIS reader</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-R1</td>
			<td>A reader of conductance and capacitance on six channels simultaneously</td>
		</tr>
		<tr>
			<td>tethaPlate cartridge assembly</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-BG</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Clamp and slide assembly jig</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-A1</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Lipid coated coplanar gold electrodes</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-T10</td>
			<td>Coplanar&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&#160;</td>
		</tr>
		<tr>
			<td>tethaQuick software</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-B1</td>
			<td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td>
		</tr>
		<tr>
			<td>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 99.9%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>pH 7</td>
		</tr>
	</tbody>
</table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

Aqui relatamos um protocolo para investigar a transferência de calor entre nanopartículas de ouro irradiadas (GNPs) e membranas lipídicas bicamadas por eletroquímica usando membranas lipídicas bicamadas amarradas (tBLMs) montadas em eletrodos de ouro. Os GNPs modificados irradiados, como os GNPs conjugados com estreptavidina, estão incorporados em tBLMs contendo moléculas-alvo, como a biotina. Usando essa abordagem, os processos de transferência de calor entre GNPs irradiados e membrana lipídica bicamada modelo com entidades de interesse são mediados por um raio laser horizontalmente focado. O modelo computacional preditivo térmico é usado para confirmar as mudanças de condutância eletroquimicamente induzidas nos tBLMs. Sob as condições específicas utilizadas, a detecção de pulsos de calor exigia fixação específica das nanopartículas de ouro à superfície da membrana, enquanto as nanopartículas de ouro não ligadas não conseguiram obter uma resposta mensurável. Esta técnica serve como um poderoso biosensor de detecção que pode ser utilizado diretamente para o design e desenvolvimento de estratégias para terapias térmicas que permitem a otimização dos parâmetros de laser, tamanho das partículas, revestimentos de partículas e composição.

O substrato de ouro sobre o qual os tBLMs podem ser criados é mostrado na Figura 1. Um esquema da configuração experimental é apresentado na Figura 2.
Os eletrodos de ouro coplanar, como mostrado na Figura 1A,são feitos de substrato de base de policarbonato de 25 mm x 75 mm x 1 mm com matrizes de ouro padronizadas. Uma camada adesiva transparente define as seis câmaras de medição individuais. O el...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Este trabalho descreve um protocolo para alcançar o monitoramento dinâmico e não invasivo da transferência de calor de nanopartículas de ouro irradiadas a laser para tBLMs. O sistema combina espectroscopia de impedância para a medição em tempo real de mudanças de condução através dos tBLMs, com um raio laser horizontalmente focado que impulsiona a iluminação de nanopartículas de ouro, para produção de calor.

Membranas lipídicas de bicamadas tethered para monitorar a transferência de calor entre nanopartículas de ouro e membranas lipídicas

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

tethered-bilayer-lipid-membranes-to-monitor-heat-transfer-between

Research

JoVE Journal

Biology

2.6K visualizações.  University of Technology Sydney. Este trabalho descreve um protocolo para alcançar o monitoramento dinâmico e não invasivo da transferência de calor de nanopartículas de ouro irradiadas a laser para tBLMs. O sistema combina espectroscopia de impedância para a medição em tempo real de mudanças de condução através dos tBLMs, com um raio laser horizontalmente focado que impulsiona a iluminação de nanopartículas de ouro, para produção de calor.

Vídeo: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Método para o isolamento de Francisella tularensis Membranas externas

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Biologia

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Gramicidina baseado em Ensaio de fluorescência; para determinar Moléculas Pequenas Potencial para Modificação de Propriedades bicamada lipídica

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

Modelagem de Membranas Biológicas, com placas de circuito e medição de sinais elétricos em Axônios: Exercícios Laboratório Student

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Cromatografia de afinidade lipídica mediada por vesículas com separação de células Magnetic Ativado (LIMACS): um novo método para analisar proteínas lipídios Interação

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstituição de um canal de Kv para membranas lipídicas de Estudos estruturais e funcionais

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Usando Caco-2 em células para estudo LIPID Transportes pelo intestino

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Isolamento e composicional Análise da cutícula vegetal lipídicas poliéster Monómeros

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Quantificação de lipídios abundância e avaliação da distribuição de lipídios em Caenorhabditis elegans por Nilo vermelho e coloração do óleo vermelho O

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Medição do metabolismo energético, no tecido da retina explantado, usando análise de fluxo extracelular

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Cotocação e gota lipídica manchando de enocitos nas larvas de Drosophila

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...