Flp in t rex cell line development

Human osteosarcoma U2OS cells expressing the tetracycline responsible element of Flp-In™ T-REx system (FT-U2OS, gift from Karmella Haynes, Arizona State University, (Haynes and Silver, 2011 (link))) were cultured in Dulbecco’s Modified Eagle’s medium without sodium pyruvate, L-Arg, and L-Lys (DMEM, Wisent) supplemented with 10% of 10 kDa cut-off fetal bovine serum (Wisent), 100 μg/mL streptomycin, 100 units/mL penicillin (Thermo), 15 μg/mL blasticidin (Wisent), and 150 μg/mL hygromycin B (Wisent). The cell media was also supplemented with 0.398 mM L-Arg, 0.274 mM L-Lys, and 3.47 mM L-Pro. The cells were grown at 37°C with 5% CO₂ in 10 cm dishes (TPP, FroggaBio) or in 6-well plates (Greiner BioOne). Following the recommendations of Flp-In™ T-REx cell line development of ThermoFisher Scientific (http://www.thermofisher.com/), we developed cell lines stably expressing the wild-type CSNK2A1-HA (WT) or the inhibitor resistant forms of double mutant (DM, V66A/I174A) of the kinase with tight tetracycline regulation. The exogenous CSNK2A1 has a C-terminal HA tag to be able to distinguish from the endogenous kinase. The cell lines were induced 48h prior to inhibitor treatment with 1 μg/mL tetracycline and were kept induced during the inhibitor treatment. Cells were challenged in two independent experiment with a range of inhibitor concentrations as indicated in the figures.