Once the cells are thawed and placed into culture, it is important to passage the cells a minimum of 2 or 3 times to ensure the growth rate of the cells is as expected.

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 subculturing the cells at low densities on multiple consecutive days.

It is always essential to establish the viable cell density when sub-culturing and then add a defined number of cells to new culture vessels to have a record of the culture health. To ensure an optimal protein yield, cell viability should always be above 95%.