Lonza's recommended plating density for primary cells is established for two main reasons: 1. To ensure residual DMSO is sufficiently diluted while cells are initially attaching and 2. to allow a maximum number of population doublings per passage. With regards to DMSO concentration, we have seen on multiple occasions that the effects of centrifugation can be significantly more damaging than the effects of residual DMSO on primary cells. As such, for most cell types, instead of centrifuging the cells, we recommend plating the cells directly at a recommended plating density. When plated at this density, the residual DMSO will not be sufficient to affect the cells and the cells will not be harmed by the effects of a centrifugation step. Most research agrees that the tolerable threshold for DMSO concentration is 1%. As most of the cells Lonza offers are cryopreserved in contain 10% DMSO, if the vial is not diluted into at least 10 ml of media initially, the residual concentration of DMSO may not be diluted sufficiently which may cause issues in viability and/or plating efficiency. Ideally, most Lonza cells should be diluted into at least 20 ml of media for initial plating. Nonetheless, initially plating cells at any density other than what is recommended on the protocol will void any product guaranties. With regards to population doublings per passage, Lonza's recommended plating density is established to ensure that the cells will be able to undergo a maximum number of population doublings per passage. While it may take longer for cells to reach confluence, cells will be able to be plated into a larger number of flasks initially and will be able to be split at a higher ratio when cultured per our recommended culture density. Plating at the recommended culture density also minimizes the number of times the cells need to be subcultured and maximizes the amount of time between subculture. As subculturing can be stressful on cells, this maintains better cell health and morphology. As most Lonza cells require at least some cell-cell interaction to proliferate, plating under Lonza's recommended plating density may result in poor proliferation or senescence.