P-glycoprotein (P-gp)/MDR1 activity assay

To determine P-glycoprotein transporter activity, at least 20 hIOs per group were used in triplicate. hIOs were placed into 4-well plates, washed three times with Hank’s balanced salt solution (HBSS with calcium and magnesium, pH = 7.4; Invitrogen) containing 25 mM HEPES and incubated at 37 °C for 30 min. The P-gp substrate paclitaxel, in DMSO (10 μM, Sigma-Aldrich), was added to hIO cultures and incubated for 2 h on a shaker at 50 r.p.m. in the presence or absence of verapamil, a P-gp inhibitor, in phosphate-buffered saline (PBS; 50 μM, Sigma-Aldrich). After incubation, hIOs were washed three times with HBSS and ruptured with an ultrasonic cell disruptor. The homogenate was centrifuged at 13,000×g for 10 min at 4 °C, and the resulting supernatant was collected for analysis. The concentration of paclitaxel in each sample was quantitated by an LC-ESI/MS/MS analysis using a 3200 QTRAP LC-MS/MS system (Applied Biosystems, Foster City, CA, USA) equipped with a Turbo V^TM Ion Spray source and an Agilent 1200 series HPLC system (Agilent Technologies).