Name: cholesterol efflux assay
Uploaded: 2012-03-06T13:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

Este trabalho foi suportado por concessões do National Saúde e Pesquisa Médica do Conselho da Austrália (NHMRC) (526.615 e 545.906) e em parte pelo Programa do Governo de Victoria da OIS. DS é um companheiro de NHMRC.

1. Preparação de [3 H]-colesterol <ol><li> Na hotte, dispensar a quantidade necessária de [3 H] colesterol em um tubo de microcentrífuga 1,5 ml (0,5 uCi (19 kBq) por poço é necessário para um ensaio típico). </li><li> Se [3 H]-colesterol é suspenso em tolueno, secá-la para baixo completamente com N2 gás e ressuspender com etanol a 100% para uma concentração final de 1 uCi (37 kBq / uL Vortex. E ​​misturar bem. </li></ol> 2. Células proteções e Rotulagem colesterol celular Este protocolo foi testado utilizando os seguintes tipos de células: monócitos humanos 4,5, THP-1 humanos monócitos-macrófagos 6,7,8, RAW 264.7 macrófagos murinos 5,9,10,11, células HeLa 12, de veia umbilical humana endotelial As células (HUVEC) e células BHK-21 9, fibroblastos humanos e rato 13,14, células HepG2 humanas hepatocarcinoma 15 e, na forma modificada, a partir de plaquetas &lt;sup&gt; 16. <ol><li> Ressuspender as células e contá-las. Células da placa em 12 poços na densidade final de 0.2x10 6 células por poço em 0,9 ml de meio completo. Células irá continuar a crescer e atingirá 0.8x10 6 células por poço por o tempo da experiência de efluxo. Para as células que não se dividem, tais como diferenciadas células THP-1, a densidade de sementeira deve ser 0.8x10 6 células por poço. Se usando 6 - ou placas de 24 poços, duplo ou metade do número de células por poço, respectivamente. O número de poços deve ser suficiente para determinações quadruplicado para cada condição experimental. </li><li> Se a diferenciação de células THP-1 é necessária, adicionar PMA (concentração final de 0,1 ug / ml) aos meios de comunicação para 48-72 h. </li><li> Adicionar uma pequena alíquota de meios para dentro do tubo de microcentrífuga contendo o [3 H] colesterol. </li><li> Lavar os lados do tubo com os meios de antes da adição de [3 H] colesterol em uma alíquota separada de meio completo (100 uL / poço). </li> &lt;li&gt; Adicionar os meios contendo [3 H] colesterol para os poços com células (volume final por alvéolo é de 1 ml). Se nenhum tratamento de células é necessária antes de rotulagem, as células podem ser colocadas em [H 3] meio contendo colesterol. </li><li> Incubar as células durante 48 horas em cultura de células incubadora (37 ° C CO, 5% 2). </li></ol> 3. Incubação de equilíbrio <ol><li> Após 48 horas de incubação, verificar células ao microscópio. Garantir que eles são saudáveis ​​e estão em confluência de aproximadamente 80%. </li><li> Remova a mídia contendo [3 H] colesterol. Lave as células cuidadosamente com PBS. Repita 3 vezes de lavagem. </li><li> Preparar meios isentos de soro. Se necessário, ativar as células adicionando agonista LXR (como TO-901317 a 1-4 mMol / L) ou AMPc (0,3 mmol / L; para células de origem do mouse apenas) para soro livre-media. </li><li> Adicionar 500 ul de soro meios de comunicação livres para cada poço. </li><li> Incubar durante 18 horas na célula incubadora de cultura (37 ° C CO, 5% 2). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Incubação efluxo do colesterol <ol><li> Após 18 horas de incubação em meio isento de soro, vá células ao microscópio. Garantir que eles são saudáveis ​​e confluente (mínimo de 80% de confluência). </li><li> Preparar uma solução de colesterol aceitadores de efluxo em meio isento de soro. Exemplos de aceitadores incluem: A apolipoproteína AI (apo AI) (concentração final 10 ug / ml) Lipoproteína de alta densidade (HDL) (concentração final 20 ug / ml) Ciclodextrina (concentração final 200 ug / ml) Plasma (concentração final de 1-2%) </li><li> Lave as células cuidadosamente com PBS. </li><li> Adicionar 250 uL de meios isentos de soro com aceitadores a cada poço. </li><li> Deixar um conjunto de poços para determinar fundo de efluxo (efluxo de mídia livre com soro NO aceitante) </li><li> Incubar as células durante 2 horas em cultura de células incubadora (37 ° C, 5% de CO 2). Duração da incubação pode variar de efluxo 30 min a 8 h, se necessário. </li></ol> 5. Processamento Samples <ol><li> Após 2 horas de incubação verificar células ao microscópio. </li><li> Colete mídia em tubos de microcentrífuga de 1,5 ml. Girar a 14.000 rpm para min 1-10 à temperatura ambiente para remover os restos celulares. </li><li> Transferir 100 uL meios em 7 ml frasco de cintilação. Adicionar 5 ml de gel de Insta-Plus (PerkinElmer) e mistura de vórtice. Armazenar amostras restantes a 4 ° C. </li><li> Colocar as placas no congelador durante 30 minutos. Adicionar 500 uL de dH2O a cada poço. Verificar as células sob microscópio para assegurar que todas as células têm levantada a partir do fundo dos poços. Se ainda aderente, deixe placas com dH 2 O a 4 ° C poços durante a noite ou arranhão. </li><li> Uma vez que todas as células têm levantado, pipetar cima e para baixo para quebrar aglomerados celulares. Transferir 100 uL em frascos de cintilação 7 ml. Adicionar 5 ml de gel de Insta-Plus (PerkinElmer) e mistura de vórtice. Armazenar placas restantes a 4 ° C. </li><li> Coloque frascos de cintilação em contador de cintilação. Configurar contador para contar para [3 H] emunidades DPM. </li></ol> 6. Analisando os resultados <ol><li> A taxa de efluxo de colesterol é geralmente expressa como uma proporção de colesterol movido a partir de células para o aceitador. A fórmula seguinte é utilizado: </li></ol><img alt="Equação 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> O efluxo específica é calculada como a diferença entre o efluxo na presença ou ausência do aceitador (branco). </li></ol><img alt="Equação 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Linha do tempo <img alt="A Figura 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Os resultados representativos Um exemplo de um resultado de uma experiência de efluxo de colesterol é mostrado na fig. 1. Nesta experiência THP-1 monócitos humanos foram diferenciadas em macrófagos e de efluxo de colesterol para aceitadores diferentes foitestado. Efluxo de colesterol e médio sem receptores (&quot;em branco&quot;) foi de 0,79% e este valor foi considerado como um efluxo não específico e foi subtraído de outros valores. Efluxo de colesterol para a saúde humana apoA-I (concentração final 30 ug / ml) foi de 4,75%. Uma amostra de plasma de referência foram incluídos nesta experiência para controlar inter-experimentais variabilidade. Uma amostra de referência pode ser utilizado para a normalização de dados através de um grande número de experiências, mas que encontramos prudente repetir o ensaio se a variabilidade é alta. Um plasma do paciente (concentração final de 2%) foi testada antes e depois de o paciente foi tratado com a medicação. Concluiu-se que neste medicação do paciente teve um impacto negativo sobre a capacidade do plasma para suportar o efluxo de colesterol. Outro exemplo de um resultado da experiência de efluxo é mostrado na fig. 2. Neste experimento RAW 264.7 de macrófagos foram ativados ou não ativados por incubação durante a noite com LXR agonista TO-901317 (concentração final1 mmol / L) e de efluxo de colesterol para a mesma amostra de plasma humano (2%) foi testado. Concluiu-se que a activação da expressão celular de transportadores ABC com agonista LXR aumenta a capacidade das células para libertar o colesterol para o aceitador extracelular. <img alt="A Figura 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Figura 1. Efluxo de colesterol a partir de células THP-1 a receptores de vários. Percentagem de efluxo de colesterol (isto é, a proporção de colesterol marcado movido a partir de células para o aceitador especificado) é mostrada após subtracção dos valores em branco. Média ± DP de determinações quadruplicado, * p &lt;0,05. <img alt="A Figura 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Figura 2. Efluxo de colesterol a partir de 264,7 Percentagem activado ou não activado com LXR agonista ao plasma humano. RAW de efluxo de colesterol (isto é, a proporção de colesterol marcado movido a partir de células àaceitador especificado) é mostrada após subtracção dos valores em branco. Média ± DP de determinações quadruplicado, * p &lt;0,001.

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Os autores declaram sem conflitos de interesses relacionados a este estudo.

O método descrito para medir o efluxo de colesterol é concebido para medir o movimento do colesterol a partir de células de um aceitador de colesterol extracelular. Há várias considerações críticas na compreensão desta metodologia. Rotulagem e de equilíbrio Na metodologia descrita colesterol marcado é adicionado ao soro contendo soro. Embora nunca investigada em detalhe, presume-se que o colesterol é incorporado nas lipoproteínas do soro e são absorvid...

<table border="1"><tbody><tr><td> Reagentes \ Células </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> De mídia completo </td><td colspan="3"> RPMI - FCS 10% - 1% de L-glutamina - Strep Pen 0,2% </td><td> M199 - FCS 5% - 1% de L-glutamina - Strep Pen 1% - HEPES 2% (1M) - 7% suplemento de crescimento de células endoteliais (ECGS) </td><td colspan="2"> DMEM - FCS 10% - 1% de L-glutamina - Strep Pen 0,2% </td></tr><tr><td> Séricos Mídia Livre </td><td colspan="3"> RPMI - 1% de L-glutamina - Strep Pen 0,2% </td><td> M199 - 1% de L-glutamina - Strep Pen 1% - HEPES 2% (1M) - Célula endotelial crescer 7%º suplemento (ECGS) </td><td colspan="2"> DMEM - 1% de L-glutamina - Strep Pen 0,2% </td></tr><tr><td> Método passagem </td><td> 1 x Tripsina </td><td> Suspensão </td><td> Raspar </td><td colspan="3"> 1 x Tripsina </td></tr><tr><td> Relação de passagem </td><td> 3:10 </td><td> 01:03 </td><td></td><td> 1:20 </td><td> 01:03 </td><td> 01:03 </td></tr><tr><td> Ativação de células (opcional) </td><td></td><td colspan="5"> TO-901.317 - LXR-agonista - Conc final. 4 mm </td></tr><tr><td> Outros reagentes </td><td></td><td> forbol 12-miristato 13-acetato (PMA) - Diferenciação celular - Conc final. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Teor de colesterol de células deve ser mantida dentro dos limites muito apertados, muito ou pouco colesterol em alguns resultados de células em ruptura das membranas celulares, a apoptose e necrose 1. As células podem colesterol fonte da síntese intracelular e de lipoproteínas plasmáticas, ambas as fontes são suficientes para satisfazer plenamente as necessidades de células para o colesterol. Os processos de síntese de colesterol e absorção são fortemente regulados e deficiências de colesterol são 2 raro. Excesso de colesterol é de 3 problema mais comum. Com a excepção dos hepatócitos e para algumas células grau adrenocorticais, as células são incapazes de degradar o colesterol. As células têm duas opções para reduzir seu teor de colesterol: para converter colesterol em ésteres de colesterol, uma opção com capacidade limitada como sobrecarga células com ésteres de colesterol também é tóxico, e efluxo do colesterol, uma opção com capacidade potencialmente ilimitado. Efluxo do colesterol é uma especificaçãoprocesso ific que é regulada por uma série de transportadores intracelulares, tais como ligação de ATP cassete transportador proteínas A1 (ABCA1) e G1 (ABCG1) e B1 eliminador de tipo de receptor. O aceitador de natural de colesterol no plasma é de lipoproteína de alta densidade (HDL) e da apolipoproteína AI. O ensaio de efluxo de colesterol é concebido para quantificar a taxa de efluxo de colesterol a partir de células cultivadas. Mede a capacidade das células para manter o efluxo de colesterol e / ou a capacidade de plasma aceitadores para aceitar o colesterol libertado a partir de células. O ensaio consiste nos passos seguintes. Passo 1: etiquetagem colesterol celular por adição de colesterol rotulados para meio contendo soro e incubando com células de 24-48 h. Este passo pode ser combinado com o carregamento de células com colesterol. Passo 2: a incubação das células em meio isento de soro para equilibrar o colesterol marcado entre todos os pools de colesterol intracelular. Esta fase pode ser combinado com a activação do celular CHtransportadores olesterol. Passo 3: a incubação de células com aceitador extracelular e quantificação de movimento do colesterol a partir de células marcadas para o aceitador. Se precursores de colesterol foram utilizados para etiquetar o colesterol recém-sintetizado, um quarto passo, a purificação do colesterol, pode ser necessária. O ensaio proporciona a seguinte informação: (i) como um tratamento particular (uma mutação, um knock-baixo, a superexpressão um ou um tratamento) afecta a capacidade da célula para o colesterol de efluxo e (ii) como a capacidade de aceitadores de plasma para aceitar o colesterol é afectada por uma doença ou um tratamento. Este método é geralmente usado no contexto de pesquisas cardiovasculares, doenças metabólicas e neurodegenerativas, infecciosas e doenças reprodutivas.

Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

Ensaio de efluxo de colesterol | Medicina | Vídeo

Cholesterol Efflux Assay

O ensaio de colesterol é concebido para quantificar a taxa de efluxo de colesterol a partir de células cultivadas e da capacidade de plasma aceitadores para aceitar o colesterol libertado a partir de células. O ensaio consiste de marcação das células com o equilíbrio do colesterol, de colesterol entre pools intracelulares e libertação de colesterol para um aceitador extracelular.

Ensaio de efluxo do colesterol

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

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29.7K visualizações.  Baker IDI Heart and Diabetes Institute. O ensaio de colesterol é concebido para quantificar a taxa de efluxo de colesterol a partir de células cultivadas e da capacidade de plasma aceitadores para aceitar o colesterol libertado a partir de células. O ensaio consiste de marcação das células com o equilíbrio do colesterol, de colesterol entre pools intracelulares e libertação de colesterol para um aceitador extracelular.

Vídeo: Cholesterol Efflux Assay

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Enriquecimento de Tecidos mamíferos e Oócitos xenopus com colesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Bioquímica

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Espectroscopia de correlação de fluorescência de variação spot para análise de difusão molecular na membrana plasmática de células vivas

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Bioquímico celular-free fluorométrica ensaio enzimático para medição do elevado-throughput de peroxidação lipídica em lipoproteína de alta densidade

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Imunologia e Infecção

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

Ensaio de Troca Lipídica em Células Vivas

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Efeitos diferenciais de drogas hipolipemiantes na modulação da morfologia das partículas de colesterol

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Medicina

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Ensaio de efluxo de colesterol Nitrobenzoxadiazole-etiquetadas do elevado-throughput

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

A proteolipossoma-Based Efluxo Ensaio para determinar as propriedades de uma única molécula de Cl<sup&gt; -</sup&gt; Canais e Transportadores

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Biologia

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 ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

Ensaio de absorção de LDL colesterol usando célula viva análise com célula de imagem monitoramento de integridade

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

O Isolamento da Linfa Mesentérica Corrente em Camundongos para Quantificar a Cinética In Vivo de Absorção de Lipídios da Dieta e Secreção de Quilomícrons

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...