| RRC ID |
36935
|
| Author |
Miyamoto R, Nozawa T, Kimura M, Shiozuka K, Tabata K.
|
| Title |
Development and validation of semiautomated 96-well transport assay using LLC-PK1 cells transfected with human P-glycoprotein for high-throughput screening.
|
| Journal |
Assay Drug Dev Technol
|
| 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.
|
| Volume |
13(2)
|
| Pages |
79-87
|
| Published |
2015-3-1
|
| DOI |
10.1089/adt.2014.621
|
| PMID |
25785771
|
| MeSH |
ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
Animals
Biological Assay / instrumentation*
Cell Line
Drug Design
Drug Evaluation, Preclinical / instrumentation*
Equipment Design
Equipment Failure Analysis
Flow Cytometry / instrumentation
High-Throughput Screening Assays / instrumentation*
Kidney / cytology
Kidney / drug effects*
Kidney / metabolism*
Robotics / instrumentation
Swine
Transfection
|
| IF |
1.187
|
| Times Cited |
3
|
|
WOS Category
|
PHARMACOLOGY & PHARMACY
BIOCHEMICAL RESEARCH METHODS
|
| Resource |
| Human and Animal Cells |
LLC-GA5-CoL150(RCB0871) |
