Este trabalho foi apoiado pelos EUA prémio Serviço Público de Saúde NS-071530.

1. Preparação de Células <ol><li> Crescer pituitárias AtT20 células em meio Dulbecco modificado por Eagle (DMEM) suplementado com soro de cavalo a 10% e manter as culturas a 37 ° C numa atmosfera humidificada de 5% de CO 2. </li><li> Manter a cultura por repicagem as células a cada 5 a 7 dias utilizando um procedimento de tripsinização padrão. </li><li> Revestimento dos poços de fundo, preto claro, placas de 96 poços com 50 ul de poli-L-lisina. Permitir que os poços para secar durante 30 min na incubadora. </li><li> Placa as células nas placas de 96 poços contendo 200 uL meios de densidade de 30.000 células por poço e armazenar as células na incubadora durante 3-4 dias. </li><li> Utilizar uma configuração de aspiração, que consiste em um frasco rolhado litros 4 ligado a um vácuo em uma extremidade e uma ponta de pipeta 200 uL na outra extremidade, para aspirar lentamente o meio de cultura a partir da monocamada de células. </li><li> Lavar as células com 200 uL da solução tampão normal (132 mM de NaCl, 5 mM de KCl, 1 mM de CaCl2, 1 mM de MgCl2, 5 mM de dextrose, 5 mM de HEPES, pH 7,4 com NaOH). </li><li> Adicionar 200 uL de solução tampão contendo a membrana corante sensível ao potencial (MPD) DiBAC 4 (3) ou HLB 021-152 (5 mM) a cada poço e incubar as células durante 40 min a 37 ° C. </li></ol> 2. Preparação de Compostos de Teste e tratamento da célula <ol><li> Prepara-se uma diluição em série dos compostos de teste (stock = 10 mM em DMSO) a partir de uma biblioteca de compostos (em formato de 96 poços), utilizando um Versette (ThermoFisher) sistema de manipulação de líquido automatizado. </li><li> Remova a placa de 96 poços, contendo os AtT20 células, a partir da incubadora e permitir que a placa para voltar à temperatura ambiente. Aspirar fora do tampão de idade e aplicar a solução de tampão fresca contendo o MPD para as células. </li><li> Usando o sistema de manuseamento de líquidos aplicar várias concentrações (10 nM a 10 uM) dos compostos de teste (2 a 20 uL) e as soluções de controlo (0,1% DMSO) (em tampão de solução containing o MPD) com a placa de 96 poços contendo as células. </li><li> Incubar as células durante 5 min com compostos de teste anteriores com as medições fluorescentes. </li></ol> 3. Ativação dos canais de GIRK, Medição e Análise Fluorescente <ol><li> Inserir a placa de 96 poços em um leitor de placas Biotek Synergy2 fluorescente e obter o sinal de fundo de fluorescência a comprimentos de onda de excitação e emissão de 520 e 560 nm, respectivamente. </li><li> Aplicar os ligandos GPCR (somatostatina = 200 nM ou carbacol = 10 uM) ou solução de controlo (tampão sozinha) (20 uL) aos poços (volume total = 220 uL) para activar os canais GIRK usando o Synergy2 sistema injector. Recolher os pontos de dados em intervalos de 10 s durante um período de 300 s de amostragem de excitação e emissão de comprimentos de onda de 520 e 560 nm, respectivamente. </li><li> Obtenha o curso do tempo e amplitude do pico da mudança fluorescente usando Biotek software Gen5 e exportar os dados para o Excel, para posterior análise. </li&gt; <li> Calcula-se a Z&#39;-factor para determinar a fiabilidade do ensaio. As placas utilizadas para Z&#39;fator de determinação contida 2 linhas cada uma positiva (ligando GPCR) e negativo (tampão sozinho) controles. O Z&#39;-factor é definido como: Z &#39;= 1 - (P + 3σ 3σ N) / | P μ - μ N |, onde P e μ μ N são os meios de controlo positivo, e sinais de controlo negativo, e s P e N σ são os desvios padrão de controlo positivo e sinais de controlo negativo, respectivamente 6. Z&#39;-factores no intervalo de 0,5 a 1,0 indicam que a qualidade do ensaio é excelente 6. Placas com Z&#39;-factores abaixo de 0,5 são excluídos da análise. </li><li> Os compostos que modulam o sinal fluorescente são novamente testados na presença de Ba 2 + ou tertiapin-Q para confirmar interior rectificador K + especificidade do canal. </li></ol> 4. RResultados epresentative Um exemplo do sinal de fluorescência medida utilizando o ensaio de canal GIRK é mostrado na Figura 2. A adição da somatostatina ligante de GPCR (200 nM) para os AtT20 células causada uma rápida, diminuição dependente do tempo no sinal fluorescente 021-152 HLB (Figura 2). Em contraste, a adição da solução de controlo produzida uma pequena subida instantânea na fluorescência que com o tempo retornado para a linha de base (Figura 2). Comparação dos valores de pico fluorescentes em placas de 96 poços de controlo e injectados com soluções de somatostatina deu um factores Z&#39;na gama de 0,5 a 0,7. Para a quantificação mais, o registo de controlo foi subtraído a partir do registo de somatostatina eo resultante somatostatina-sensível sinal analisado (Figura 2). O ensaio proporciona um método para identificar rapidamente drogas que modulam canais GIRK. Por exemplo, as drogas que inibem o cha GIRKnnel deve reduzir GPCR ligando mediadas diminuições na fluorescência, impedindo K + efluxo a partir das células. Como mostrado na Figura 3, o tratamento das células com tertiapin-Q, uma toxina que bloqueia os canais GIRK 7, produziu uma inibição da mudança de somatostatina mediada fluorescente. Propafenona, uma droga anti-arrítmica que bloqueia os canais GIRK no coração 8, também inibiu a mudança fluorescente (Figura 4). O ensaio também pode ser útil para a identificação de activadores do canal de GIRK. No entanto, a aplicação de etanol (100 e 200 mM), um activador do canal GIRK 2,3, não causou alteração significativa no sinal fluorescente em relação ao controle de solução (p&gt; 0,5). <img src="/files/ftp_upload/3850/3850fig1.jpg" alt="A Figura 1"/> Figura 1. O delineamento experimental do ensaio canal GIRK fluorescente. AtT20 células são incubadas em tampão contendo um Membrane corante fluorescente sensível potencial-(D). As moléculas de corante entrar nas células e tornar-se fluorescente após ligação a proteínas intracelulares (painel superior). A ligação de somatostatina (SOM) para a sua GPCR estimula a proteína inibidora de G (G i) causando a activação do canal de GIRK (painel inferior). O efluxo subsequente de K + para fora das células faz com que o potencial de membrana a tornar-se mais negativa e as moléculas de corante para sair das células. Como resultado, as diminuições de sinal fluorescente (painel inferior). <img src="/files/ftp_upload/3850/3850fig2.jpg" alt="A Figura 2"/> Figura 2. Representativas HLB 021-152 sinal fluorescente medido ao longo do tempo nas células AtT20 durante a adição de somatostatina ou solução de controlo. A relação da intensidade de fluorescência (F / F O) foi calculado dividindo o sinal na presença (F) da somatostatina (ou solução de controlo) pelo sinal de linha de base medido antes (F O) addição de somatostatina (ou solução de controlo). Cada ponto representa a média ± SE obtido em 5-6 poços. Somatostatina ou solução de controlo foi adicionado no tempo zero (↓). A adição da solução de controlo causou um aumento pequeno transiente no sinal fluorescente. Isto resultou de uma mudança de temperatura causada por injecção da solução. <img src="/files/ftp_upload/3850/3850fig3.jpg" alt="A Figura 3"/> Figura 3. Tratamento das células com AtT20 tertiapin-Q (500 nM) inibe a diminuição da somatostatina mediada no sinal fluorescente e ligando-se a bloquear os canais de GIRK. Cada ponto representa a média ± SE obtido em 4-6 poços. Somatostatina foi adicionado no tempo zero (↓). <img src="/files/ftp_upload/3850/3850fig4.jpg" alt="A Figura 4"/> Figura 4. Tratamento das células com AtT20 propafenona (20 uM) também inibe a diminuição da somatostatina mediada-ino sinal fluorescente. Cada ponto representa a média ± SE obtido em 5-6 poços. Somatostatina foi adicionado no tempo zero (↓).

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Enquanto potencial de membrana sensíveis corantes fluorescentes foram usados ​​para identificar drogas que modulam canais iônicos 9,10, este é o primeiro relato de sua aplicação para neuronal GIRK droga Discovery Channel. O ensaio GIRK canal fluorescente apresentado aqui fornece um método rápido, confiável e em tempo real para o rastreamento de ligante fechadas canais de K +. O ensaio pode ser modificadas para utilização com uma vasta gama de células incluindo linhas de células imor...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10-013 </td></tr><tr><td> De soro de cavalo </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> Placas de 96 poços </td><td> Corning </td><td> 3603 </td></tr><tr><td> Poli-L-lisina </td><td> Sigma-Aldrich </td><td> P4707 </td></tr><tr><td> Somatostatina </td><td> Sigma-Aldrich </td><td> S9129 </td></tr><tr><td> Carbacol </td><td> Sigma-Aldrich </td><td> C4382 </td></tr><tr><td> HLB 021-152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> Versette manipulador de líquidos automatizado </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 leitor de placas fluorescentes </td><td> Biotek </td><td></td></tr><tr><td> Gen5 análise software </td><td> Biotek </td><td></td></tr></tbody></table> Tabela 1. Tabela de reagentes específicos e equipamentos.

a fluorescent screening assay for identifying modulators of girk channels

Proteína G-gated para dentro do rectificador K + (GIRK) canais de funcionar como mediadores celulares de uma vasta gama de hormonas e neurotransmissores e são expressos no cérebro, coração, músculo esquelético e 1,2 tecido endócrino. Canais GIRK tornar-se activado após a ligação de ligandos (neurotransmissores, hormonas, fármacos, etc) para a sua plasma ligada à membrana, acoplados à proteína G (GPCRs receptores). Esta ligação faz com que a estimulação de proteínas G (G i e G o) que, subsequentemente, se ligar e activar o canal GIRK. Uma vez aberto o canal GIRK permite a circulação de K + para fora da célula fazendo com que a membrana em repouso potencial para se tornar mais negativo. Como conseqüência, GIRK ativação de canais em neurônios diminui a formação de potencial espontâneo de ação e inibe a liberação de neurotransmissores excitatórios. No coração, a activação do canal de GIRK inibe a actividade pacemaker assim abrandar o ritmo cardíaco. <pclass = &quot;&quot; jove_content canais&gt; GIRK representar novos alvos para o desenvolvimento de novos agentes terapêuticos para tratamento da dor neuropática, a toxicodependência, arritmias cardíacas e outros distúrbios 3. No entanto, a farmacologia destes canais permanece largamente inexplorado. Embora um número de drogas, incluindo agentes anti-arrítmicos, fármacos antipsicóticos e antidepressivos bloquear o canal GIRK, esta inibição não é seletivo e ocorre em concentrações relativamente elevadas de drogas 3. Aqui, descrevemos um teste de triagem em tempo real para identificar novos moduladores de canais GIRK. Neste ensaio, as células neuronais AtT20, expressando canais GIRK, são carregados com potencial de membrana sensíveis corantes fluorescentes, tais como bis-(1,3-dibutylbarbituric ácido) trimethine oxonol [DiBAC 4 (3)] ou HLB 021-152 (Figura 1 ). As moléculas de corante se fortemente fluorescente absorção seguinte para dentro das células (Figura 1). Tratamentodas células com ligandos GPCR estimula os canais GIRK para abrir. A resultante de K + de efluxo para fora da célula faz com que o potencial de membrana a tornar-se mais negativa e do sinal fluorescente para diminuir (Figura 1). Assim, as drogas que modulam a K + efluxo através do canal GIRK pode ser ensaiada utilizando um leitor de placa fluorescente. Ao contrário de outros íons ensaios canal de rastreio, espectrometria de absorção atômica, tais 4 ou análise radiotraçador 5, o canal fluorescente GIRK ensaio fornece um procedimento de triagem rápida, em tempo real e barato.

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Um procedimento de triagem em tempo real para identificação de drogas que interagem com a proteína G-gate dentro retificador K<sup&gt; +</sup&gt; (GIRK) canais é descrito. O ensaio utiliza potencial de membrana sensíveis corantes fluorescentes para medir GIRK actividade do canal. Esta técnica é adaptável para utilização num certo número de linhas celulares.

Um ensaio de rastreio fluorescente para identificação Moduladores de Canais GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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Research

JoVE Journal

Biology

14.3K visualizações.  University of South Carolina, School of Medicine. Um procedimento de triagem em tempo real para identificação de drogas que interagem com a proteína G-gate dentro retificador K + (GIRK) canais é descrito. O ensaio utiliza potencial de membrana sensíveis corantes fluorescentes para medir GIRK actividade do canal. Esta técnica é adaptável para utilização num certo número de linhas celulares.

Vídeo: A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Biologia

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Armadilha cromossomo associados para Identificar Interações de longo alcance DNA

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Alto rendimento Triagem e Biossensoriais com Fluorescente C. elegans Cepas

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

A conversão de um ELISA de captura para um ensaio de xMAP Luminex usando um método de rastreio de anticorpos Multiplex

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

High-throughput screening para pequenas moléculas Moduladores de canais de potássio Interiores retificador

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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 ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Barcoding Genetic com proteínas fluorescentes para Aplicações Multiplexados

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

Avaliação da Função Renal Zebrafish Usando um teste de eliminação fluorescente

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...