Dynamics of Cell Cycle Regulators: Artefact-Free Analysis by Recultivation of Cells Synchronized by Centrifugal Elutriation

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Abstract
Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.