Slide a lyzer g2 10k dialysis cassettes

Constructs were transiently transfected in HEK293-EBNA1-6E cells (NRC) or Expi293F cells (Thermo Fisher) at a density of 1.2-1.6 million cells/mL or 3 million cells/mL, respectively, using 25 kDa linear polyethylenimine (PEI) at a 3:1 PEI:DNA ratio in OptiMEM reduced serum medium (Gibco), with a heavy chain: light chain ratio of 1:1. HEK293-EBNA1-6E cells were maintained in Freestyle 293 Expression Medium (Gibco) and were supplemented with 0.5% tryptone 24-36 hours after transfection. Expi293F cells were maintained in Expi293 Expression Medium (Thermo Fisher). Cell supernatants were harvested after 96 hours and filtered through 0.45-μM filters (Millipore). Antibodies were purified via HiTrap Protein A HP columns (GE Healthcare) run at a flow rate of 0.5-1 mL/min on Äkta Start protein purification system (GE Healthcare). Antibodies were eluted using 0.1M glycine pH 2, dialyzed with 3 changes of PBS pH 7.4 using Slide-A-Lyzer-G2 10K dialysis cassettes (Thermo Fisher), and concentrated using 30,000 kDa molecular weight cutoff polyethersulfone membrane spin columns (Pierce). Final concentrations of purified antibodies were quantified with Nanodrop 2000 (Thermo Fisher).