The recommended cell number will vary depending on which Optimized protocol is being used. In general, using less than 2x10exp5cells per reaction causes a major increase in cell mortality. For some cell lines we have tried cell numbers up to 3x10exp7. The problem here is that you end up with a very large cell pellet leading to a dilution of the nucleofector solution as the maximum cuvette volume is 100µL to 110µl. We suggest increasing the cell number successively from experiment to experiment but not exceeding a cell pellet volume of 50 µl.