After transfection complexes are formed, they need to be incubated with cells to facilitate the transfer of the nucleic acids into the cells. Incubation allows the complexes to interact with the cell membrane and enter the cells through various mechanisms, including endocytosis and direct penetration.

The duration and conditions of incubation can vary depending on the type of transfection complex and the specific cell type being used. Typically, cells are incubated with the transfection complex for several hours, up to 24-48 hours. The temperature and CO2 concentration of the incubation environment should also be optimized for the specific cell type being used.

It is important to note that during incubation, the transfection complexes can be vulnerable to degradation by enzymes and other factors in the cell culture medium. To prevent this, some transfection protocols recommend adding serum-free medium or other supplements to the culture medium during incubation.

Once the incubation period is complete, the cells can be washed to remove any residual transfection complexes and then returned to normal culture conditions to allow for expression or analysis of the transfected nucleic acids.