The cell number is very much dependent on the cell type you use. For high throughput Nucleofection®, we usually start with one fifth of the optimal amount recommended for the standard Nucleofection® in the 100µl cuvette. Currently the absolute cell number per well ranges from 104 and 106 cells. The lower limits are often dictated by the minimum amount of cells required for optimal plating density on 96-well cell culture plates so that one may expect that even lower cell numbers might work in some settings.