title: Development and validation of semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-glycoprotein for high-throughput screening.
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.
authors.affiliation: 1 Drug Metabolism and Pharmacokinetics Research Division, Astellas Research Technologies Co., Ltd. , Ibaraki, Japan .
authors.forename: Rei
authors.initials: R
authors.lastname: Miyamoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Nozawa
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kimura
authors.forename: Koichi
authors.initials: K
authors.lastname: Shiozuka
authors.forename: Kenji
authors.initials: K
authors.lastname: Tabata
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay / Q000295:instrumentation*; D002460:Cell Line; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation; D057166:High-Throughput Screening Assays / Q000295:instrumentation*; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D012371:Robotics / Q000295:instrumentation; D013552:Swine; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1