Expifectamin 293 reagent

The wild type VAR2CSA NF54 and VAR2CSA FCR3 were expressed in Expi293 (Thermo Fisher) cells according to the manufacturer’s protocols. In brief, the cells were grown shaking at 37°C and 8% CO₂, maintaining cultures at continuous log phase growth (3.0–5×10⁶) for 3–4 passages after thawing. The day before transfection, 500 mL of culture was seeded at a density of 2.5–3×10⁶ cells/mL in a 2 L flask. The day of transfection, cells were diluted back to 2.5–3×10⁶ prior to transfection. The plasmid DNA was diluted with 25 mL of Opti-MEM I medium (Thermo Fisher) to a final concentration of 1μg/mL.
Then,1.4ml ExpiFectamin^™ 293 Reagent (Thermo Fisher) was diluted with 25 ml Opti-MEM I medium, gently mixed and incubated at room temperature for 5 minutes. The diluted ExpiFectamine^™ 293 Reagent was then added to the diluted plasmid DNA, mixed by swirling, and incubated at room temperature for 20 minutes. The mixture was added to the cells slowly while swirling the flask. The flask was returned to the incubator at 37°C and 8% CO₂. After 20 hours of incubation, ExpiFectamine^™ 293 Transfection Enhancer 1 (Thermo Fisher) and ExpiFectamine^™ 293 Transfection Enhancer 2 (Thermo Fisher) were added to the transfection flask.