Quality Control of Centrifugal Elutriation for Studies of Cell Cycle Regulations

摘要

An essential and probably primary task in the study of cell cycle regulations is the synchronization of cells into specific cell cycle phases. Most of the information on cell cycle-related processes has been gained in the past through the use of chemicals or growth restriction conditions to synchronize cells. Logarithmically growing cells can alternatively be separated by centrifugal elutriation, which does not require an arrest of the cell cycle to obtain synchronized cells. By this method, cells are separated on the basis of size and, to a lesser extent, density under moderate conditions, making it ideal for isolating populations of cells in specific phases of the cell cycle with minimal metabolic perturbations. Centrifugal elutriation has alreadybeen used in several different studies of cell cycle-dependent parameters, such as the regulation of cyclins and their associated kinases or E2F-dependent transcription. However, the number of cell types separated by centrifugal elutriation for cell cycle analyses described so far is limited, and a detailed investigation of the repro-ducibility of this method to analyze cell cycle parameters in different cell types is missing. We describe data that should help investigators to obtain reproducible results on cell cycle regulations in a wide variety of different cell types using the approach of centrifugal elutriation: (i) analyzing a wide variety of mammalian cell types, we found that the quality of separation by centrifugal elutriation must reach a very specific level to enable one to properly study cell cycle regulations; (ii) we have optimized the centrifugal elutriation conditions to obtain highest quality of separation for 22 different primary cells and cell lines, focusing on those that are frequently used in investigations of cell cycle regulations; and (iii) our data allow general conclusions that could, help optimize the conditions for elutriation of other cells.