Maintaining Kv2.1-CHO and BFP-CHO Cell Lines

All CHO-K1 cell lines were maintained in polystyrene dishes (BioLite, Thermo) at 37 °C in a 5% CO₂ atmosphere in Ham’s F-12 media (Corning) containing 10% v/v FBS (Gibco), and penicillin–streptomycin (Sigma). A CHO-K1 cell line stably transfected with rat Kv2.1 (Kv2.1-CHO cells)^{56 (link)} was cultured with 1 μg/mL blasticidin and 25 μg/mL zeocin to retain stably transfected vectors. To induce expression of Kv2.1, minocycline (Enzo Life Sciences) from a stock of 2 mg/ml in 70% EtOH was added to cell culture media to a final concentration 1 μg/mL. Time of incubation with minocycline was chosen to induce a desirable amount of channel expression: 1.5 – 2 h for K⁺ conductance experiments, ~48 h for imaging and voltage clamp spectroscopy experiments. The CHO-K1 cell line stably transfected with BFP (BFP-CHO cells)^{23 (link)} expresses the blue fluorescent protein variant EBFP2 with a nuclear localization sequence.^{57 (link)} BFP-CHO cells were maintained in media containing 100 μg/mL of G418.