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.