*5.6. Animals*

Animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Kentucky (protocol no.: 2014–1233, PI: Hartz; approved April, 2014) and were carried out in accordance with AAALAC regulations, the US Department of Agriculture Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals of the NIH.

Male P-gp knockout (KO) mice (CF-1 strain; CF1-Abcb1amds—PGP) and corresponding male CF-1 wild-type (WT) mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Mice were 9 weeks old with an average body weight of 33.7 g (31–35 g) for WT mice and 36.3 g (33–42 g) for P-gp KO mice. All mice were single-housed and kept under controlled environmental conditions (21 ◦C; 51–62% relative humidity; 12-h light/dark cycle) using an Ecoflo Allentown ventilation system (Allentown Inc., Allentown, NJ, USA). Animals were monitored at least once a day and had free access to tap water and Harlan Teklad Chow 2918 rodent feed (Harlan Laboratories Inc., Indianapolis, IN, USA). After shipping, animals were allowed to acclimate to their new environment for at least 7 days prior to experiments.
