Name: cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3
Uploaded: 2011-08-17T12:00:00
Description: Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Agradecemos a drs. John Hancock, Ray Grill, Andrew Morris e Olga Chumakova pelo seu apoio. O FlexStation 3 sistema faz parte do Mecanismo de imagem UTHSC Cytodynamic. Agradecemos também os drs. Ardem Patapoutian e Michael Bandell do Scripps Research Institute e Instituto de Genômica da Novartis Research Foundation (GNF) para fornecer a linha de células-HEK293 hTRPA1 estável. A pesquisa é financiada em parte por concessões do NIH (GM081658 para MXZ) e Texas Medical Center Digestive Diseases Center (para HH) e Missão Connect / TIRR Foundation (para HH).

1. Preparações de células em placas de 96 poços Para HEK293 células transitoriamente trasfected com TRPA1 humana cDNA <ol><li> HEK293 células são cultivadas para 1-2 dias em meio mínimo essencial de Dulbecco (DMEM), contendo 4,5 glicose mg / ml (Thermo Scientific, SH3002201), suplementado com inativado pelo calor de 10% de soro fetal bovino (Invitrogen, 16000-044), 50 unidades / ml de penicilina e estreptomicina 50 mg / ml (Invitrogen, 15140-163). No dia da transfecção, ...

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Ca 2 + intracelular desempenha um papel importante em muitas condições fisiológicas e patológicas, como a liberação de neurotransmissores (3), potencial de ação cardíaco (4), a contração da célula muscular (5), transdução de sinal celular (6-7), e morte celular excitotoxic ( 8). Medição de Ca 2 + intracelular níveis ajuda a elucidar as alterações funcionais de Ca 2 +-permeável canais na membrana plasmática ou organelas intracelulares ea contribuição de Ca <s...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> FlexStation 3 </td><td> Os dispositivos moleculares </td><td> FLEX3 </td><td></td></tr><tr><td> 96-Bem, Pipetar Dicas FlexStation </td><td> Os dispositivos moleculares </td><td> 9000-0912 </td><td></td></tr><tr><td> Placas de 96 poços </td><td> Greiner Bio-one </td><td> 655090 </td><td></td></tr><tr><td> 96-Bem Limpar plano placas inferior </td><td> Costar </td><td> 3595 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12430-104 </td><td></td></tr><tr><td> Opti-MEM </td><td> Invitrogen </td><td> 31985-070 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-019 </td><td></td></tr><tr><td> Fluo-04:00 </td><td> Invitrogen </td><td> F14202 </td><td></td></tr><tr><td> PluronicF-127 </td><td> Invitrogen </td><td> P3000MP </td><td></td></tr><tr><td> Poli-L-ornitina </td><td> Sigma </td><td> P36551 </td><td></td></tr><tr><td> probenecid </td><td> Sigma </td><td> P8761 </td><td></td></tr><tr><td> Albumina de soro bovino </td><td> Sigma </td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

FlexStation Dispositivos Molecular &#39;3 é uma bancada modo multi-leitor de microplacas capaz de medição de fluorescência automatizado em multi-lamelas. É ideal para empresas de médio a alto rendimento telas em ambientes acadêmicos. Ele tem um módulo de transferência integrada de fluido equipado com uma pipeta multi-canal ea máquina lê uma coluna de cada vez para monitorar as mudanças de fluorescência de uma variedade de reagentes fluorescentes. Por exemplo, FlexStation 3 tem sido usado para estudar a função de Ca 2 +-permeável canais iônicos e G receptores acoplados à proteína, medindo as mudanças de livre intracelular Ca 2 + níveis. Transiente potencial receptor (TRP) canais são uma grande família de canais de comunicação não-seletivos que desempenham papéis importantes em muitas funções fisiológicas e fisiopatológicas. A maioria dos canais de cálcio TRP são permeáveis ​​e induzir o influxo de cálcio na ativação. Neste vídeo, demonstramos a aplicação de FlexStation 3 a estudar o perfil farmacológico do canal TRPA1, um sensor molecular para inúmeros estímulos nocivos. HEK293 células transitoriamente ou expressando estavelmente humana TRPA1 canais, cultivadas em placas de 96 poços, são carregados com um Ca 2 + sensíveis ao corante fluorescente, Fluo-4, e em tempo real as mudanças de fluorescência nessas células são medidos antes e durante a aplicação de um agonista TRPA1 usando o modo FLEX do FlexStation 3. O efeito de um antagonista TRPA1 putativa também foi examinada. Dados são transferidos do software Pro SoftMax para construir relações concentração-resposta de TRPA1 ativadores e inibidores.

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Este vídeo fornece um protocolo detalhado para o estudo do perfil farmacológico dos direitos humanos TRPA1 canais usando FlexStation 3. O protocolo abrange detalhes de preparação das células, carregando corante e operação do leitor de microplacas, FlexStation 3.

Baseados em células de Ensaio de cálcio para Médio a High Throughput Screening de Funções TRP usando FlexStation Canal 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

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