Dmem f 12 with hepes

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).

HK-2 cells derived from human proximal tubular cells were cultured in DMEM/F12 with HEPES (GIBCO, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) in a 37 °C, humidified incubator with 5% CO₂.