At the time of transfection, ExpiCHO-S cells should be in mid-log phase growth, have high viability (95% or greater) and should be diluted with fresh ExpiCHO expression medium to a final density of 6 x 10⁶cells/mL.

Once the cells are transfected, flasks should be placed back in the 37 °C incubator with 8% CO₂overnight, until the addition of the ExpiCHO Feed and ExpiFectamine CHO Enhancer, 18-22 hours post-transfection.