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| | In Vitro ATPase Assay |
| Description: | To assess the inhibition of the P-glycoprotein (Pgp/MDR1) transporter, an in vitro ATPase assay from Cyprotex was used. MDR1MDCK cells obtained from the NIH (Rockville, Md., USA) were used. Following culture, the monolayers were prepared by rinsing both basolateral and apical surfaces twice with buffer at pH 7.4 and 37 C. Cells were then incubated with pH 7.4 buffer in both apical and basolateral compartments for 40 min at 37 C. and 5% CO2 with a relative humidity of 95% to stabilise physiological parameters. For the apical to basolateral study (A-B), buffer at pH 7.4 was removed from the apical compartment and replaced with loperamide dosing solutions before being placed in the companion plates. The solutions were prepared by diluting loperamide in DMSO with buffer to give a final loperamide concentration of 5 uM (final DMSO concentration adjusted to 1%). The fluorescent integrity marker Lucifer yellow was also included in the dosing solution. |
| Affinity data for this assay | |
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