N acetylcysteine

To prepare single Lgr5 cells, crypts from Lgr5e-creERT2 mouse were isolated by incubating the opened lengthwise small-intestine pieces in the Gentle Cell Dissociation reagent (STEMCELL Technologies) on a rocking platform, at 20 rpm for 15 min at RT. Next the tissue pieces were resuspend in 10 ml ice-cold PBS containing 0.1% BSA and pipette up and down to strip off the crypts. The suspension was filtered through a 70-μm cell strainer. Filtered crypts were resuspended in 5 ml of TryPLE Express (Gibco) supplemented with Y27632 (10 μM) (Sigma-Aldrich). Crypts were transferred into a C-tube and dissociated by pre-programmed GentleMACS (Miltenyi Biotec). Cell suspension of the dissociated crypts were centrifuged and resuspended in 1 ml ice-cold DMEM/F-12 with HEPES (15 mM) (Gibco/Life Technologies) supplemented with N-acetylcysteine (0.5 mM) (Sigma-Aldrich), Y27632 (10 μM) and 1% (wt/vol) BSA. The cell number was counted by a cell counter to obtain a concentration of 1–5 × 10⁶ cells/ml. Cells were stained with 7-AAD (BD Biosciences) and Annexin V (BioLegend). Lgr5 cells were sorted through a 100-μm nozzle using BD FACS Aria III (BD Bioscience) into DMEM/F-12 with HEPES (15 mM), supplemented with N-acetylcysteine (0.5 mM) and Y27632 (10 μM).

Mouse liver microsomes (MLMs), human liver microsomes (HLMs), recombinant human P450 enzymes, NADPH-regenerating system, glutathione (GSH), N-acetyl cysteine (NAC), and N-acetyl lysine (NAL) were purchased from BD Biosciences (Bedford, MA, USA). LIM (purity >98%) was obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Acetonitrile (ACN), methanol (MeOH), and formic acid (FA) of LC/MS grade were obtained from Fisher Scientific (Pittsburgh, PA, USA). Water was purified with a Milli-Q system (Millipore, Bedford, USA) and was passed through a 0.22 μm membrane filter before use.