Although there are a few generalized rules that can be applied, the optimum number of cells to seed per well needs to be determined for each cell line studied. This is due to differences in each cell line’s size, morphology and doubling rates.
The classic initial study should include varying the number of cells seeded per well (i.e. from 1×104 to 1×105) and allowed to grow until the expected end of study (i.e. 3-5 days post-transfection) to ensure confluency is not reached in non-treated cells. Keep in mind that although the ratios are known between the surface area of a single well in a 96-plate and a well in a 6-well plate, simply multiplying by the ratio does not always work. It is best to perform the initial study to determine optimal cell number to seed in certain size plate that is being used for transfection experiment.