Primary human hepatocytes
Primary human hepatocytes were seeded on 384-well plates as described
previously to stimulate re-acquisition of in vivo physiologic
activity (10). At day 3 post seed, ivermectin (10 µM) was added to each
well. There were no media changes and no subsequent addition of
ivermectin. 40 µL of media was collected from five individual wells at
24 hours and pooled in a microcentrifuge tube (total volume 200 µL). 40
µL of media was mixed with cold acetonitrile (160 µL), vortexed for 10
min at ambient temperature, and centrifuged at 10,000×g for 15
minutes at 4°C (triplicate extractions). The supernatant was collected,
evaporated in speed vacuum, and kept frozen at -80°C until LC-MS/MS
analysis.