| Assay Detail |
MDCKII-MDR1 cells were obtained from NKI and maintained at 37°C in 5% CO2 and 95% RH using DMEM supplemented with 10% v/v FBS, 1% v/v l-glutamine, 200 μg/ml penicillin, and 100 μg/ml streptomycin sulfate. Stock flasks were split 1:10 every 3-4 days. Cells were plated for experiment at 50,000 cells/cm2 in 12-well Transwell inserts. Cell culture medium was changed on days 3 and 5 (0.5mL apical, 1.5 mL basolateral), and Permeability was measured on day 6.
Culture inserts were rinsed with Dulbecco's PBS + 10mM HEPES, pH 7.4, and then conditioned with 10uM cyclosporin for 30 min at 37°. Liquid volumes in the apical and basolateral compartments were 0.5mL and 1.0 mL, respectively, throughout the assay. Cells were incubated 5uM test compound at 37°C with constant nutation (BD Clay Adams, VWR). After 60 minutes, the cell inserts were removed and samples taken from both apical and basolateral chambers. Cells were rinsed with cold buffer and extracted with 500uL MeOH. Samples were combined 3-in-1 and analyzed by LC/MS/MS with methods individually optimized for each cassette. Permeability was determined by the equation: Pe =(V/A)*(M/t) where V = volume in the apical compartment (mL), A = surface area of the filter area (1.13cm^2), M = change in mass in the basolateral compartment, and t = time (seconds). Permeability was measured in both directions -basolateral to apical and apical to basolateral. The apparent passive permeability was calculated as the average value across the two directions.
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