Human KCC2 Protein Expression in HEK293 Cells

The various human KCC2 constructs were transfected into HEK293 cells using the MaxCyte STX electroporation. Expression of human KCC2 was confirmed by Western-blot using Rabbit anti-KCC2 as primary antibody (Sigma/Millipore #07-432). Culture media was DMEM (Sigma, D6546) with 10% FBS and 1% GlutaMAX (Gibco, 35050-038). For large scale production of protein for binding studies, suspension Expi293F cells were cultured in Expi293F media and transfected at high density with KCC2 plasmid DNA diluted to 1 mg/ml using PEIMax at 1 mg/ml^{38 (link)}. Transfection was carried out on 250 ml of suspension cells at 4 × 10⁶/ml in vented 2 L capacity roller bottles shaken in an atmosphere of 5% CO₂ at 37 °C and 140 rpm in an orbital incubator (25 mm orbit). DNA and PEIMax were pre-incubated in E293F media for 15 minutes before adding to the cells. Twenty-four hours post transfection, cultures were fed with 50% media making 500 ml total volume per roller bottle and incubated for a further 48hrs. Cell pellets were harvested 72hrs post transfection by centrifugation at 10,000 × g for 20 min and stored at −80 °C prior to purification. Cells and media were purchased from ThermoScientific; 40 kDa linear PEI Max as hydrochloride salt (catalogue #24765) from Polysciences. Mouse KCC2 was expressed in HEK293 and N2a cells as previously described^{5 (link),39 (link)–41 (link)}.