Cellular uptake study
The cellular accumulation of OXA was evaluated according to a previously
reported method with minor modifications (Ma & Yang et al., 2017). The
cells were seeded in 12-well plates (for mock and transporter
transfected cells) or 6-well plates (for DRG cells) (Corning Costar, NY,
USA) and cultured to ~90% confluence. MDCK-hOCTN1,
MDCK-hOCT2, MDCK-hOCTN2, mock, and primary DRG cells were pre-incubated
with HBSS at 37 °C for 5 min, while MDCK-hMRP2 cells were incubated for
30 min. Next, 0.5 mL of HBSS containing 40 μM OXA with or without L-THP
was added to initiate cellular uptake. The experiment was performed in a
humidified air/CO2 incubator (5% v/v) at 37 °C for 5
min (for uptake transporters) or 1 h (for efflux transporter MRP2) and
terminated by removing the incubation buffer rapidly. Next, the cells
were quickly rinsed three times with ice-cold HBSS and lysed using 100
μL of 0.01% SDS.