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Cells used for transient protein expression should be meticulously maintained from the time of thaw throughout their useful lifespan. Deleterious cell culture conditions can negatively impact not just your next transfection, but all transfections from that point forward. Cells should be well-characterized in terms of their growth rates and log phase growth range and should typically be split at mid log phase growth or slightly lower densities; do not let your cells overgrow and do not “over split” your cells by sub culturing the cells at low densities on multiple consecutive days. At the time of transfection, CHO cells, like most other cell types, should have high viability (95% or greater) and should be in low to mid log phase growth. Thus, for a CHO cell line with mid log phase at 4×10^6 viable cells/mL, transfecting cells when they reach a density of approximately 1-4×10^6 would be reasonable, with 2-3×10^6 likely being an optimal target density at the time of transfection.