Establishing Stable IMCD3 Flp-In Cell Lines

Mouse renal epithelial Flp-In cells from the inner medullary collecting duct (IMCD3 Flp-In; kind gift from M.V. Nachury) were cultured at 37°C and 5% CO₂ in DMEM/F12, HEPES (Life Technologies) complemented with 10% fetal bovine serum (FBS), and 1% L-glutamine. Stable cell lines were generated as previously described (Sang et al., 2011 (link), Torres et al., 2009 (link)). In short, the parental IMCD3 Flp-In cell line contains a stably integrated FRT cassette and was co-transfected with pOG44 coding an FLP recombinase, and the appropriate construct cloned into pgLAP5 vector (Addgene), coding for a C-terminal S- and GFP-tag, using Lipofectamine 2000 (Life Technologies). Selection by supplementing the media with 200 μg/ml hygromycin (Merck) for successful stable genomic integration was carried out, and expression of the GFP fusion protein was checked by western blot using an anti-GFP antibody (1:500; Santa Cruz Biotechnology).