| Assay Method Information | |
| | Caco-2 cells assay |
| Description: | To determine potency of compounds on inhibition of transporters, bi-directional transport studies are performed in Caco-2 cells (American Type Culture Collection, Manassas, Va.) at 37° C. in air, according toXia et al. (Expression, localization and functional characteristics of breast cancer resistance protein in Caco-2 cells. Drug Met. Disp., 33 (5): 637-643 (2005)). Prior to each experiment, the confluent cell monolayers on Transwell inserts are washed and equilibrated for 30 minutes with transport media (Hank's balanced salt solution (HBSS) containing 10 mM of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) and 10 mM of glucose, pH 7.4). The experiment is initiated by adding a solution containing the 3H-digoxin (25 nM, substrate for p-gp), or 3H-E3S (17 nM, substrate for BCRP) to either the apical (for A-to-B transport) or basolateral (for B-to-A transport) compartment in the absence or presence of various concentrations of Compound (I-1) or Ko143. At preset time points, 0.05 mL aliquot of receiving solutions are sampled from the basolateral side (for A-to-B transport) or from the apical side (for B-to-A transport), and replaced immediately with an equal amount of fresh transport media except at the last time point (the end of the incubation). The radioactivity in each sample is measured by 1450 MicroBeta TriLux, a microplate scintillation and luminescence counter (PerkinElmer Life Sciences). Radioactivity (3H) of the dosing solution is measured and used to calculate the initial donor concentration of the substrate. |
| Affinity data for this assay | |
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