Development and validation of semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-glycoprotein for high-throughput screening.
Assay Drug Dev Technol
; 13(2): 79-87, 2015 Mar.
Article
em En
| MEDLINE
| ID: mdl-25785771
ABSTRACT
Transport assays using P-gp-expressing cell lines are commonly used to identify P-gp substrates and inhibitors in drug discovery. The P-gp cell-based assay is performed manually in 12- or 24-well plates and requires improvement for high-throughput screening. In this study, we established an efficient semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-gp. The protocol was optimized with a microplate washer for exchanging media and buffer to enhance throughput. P-gp substrates and inhibitors, and the paracellular marker Dextran Texas Red® were used to validate the 96-well transport assay. Cell monolayer integrity after washing by a microplate washer was confirmed by measuring paracellular permeability of Dextran Texas Red. Permeability and net flux ratio of the P-gp substrates and the inhibitory potency of the P-gp inhibitors were comparable in 24- and 96-well plates. The regression value of net flux ratio of P-gp substrates was high between the two formats (r²=0.99). The optimized 96-well transport assay using the microplate washer was found to be an efficient high-throughput screening tool that provided the same quality data as the 24-well plate for the identification of P-gp substrates and inhibitors in drug discovery.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bioensaio
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Membro 1 da Subfamília B de Cassetes de Ligação de ATP
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Avaliação Pré-Clínica de Medicamentos
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Ensaios de Triagem em Larga Escala
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Rim
Tipo de estudo:
Diagnostic_studies
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Evaluation_studies
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Screening_studies
Limite:
Animals
Idioma:
En
Revista:
Assay Drug Dev Technol
Ano de publicação:
2015
Tipo de documento:
Article