Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication.

We report an optimised centrifugal counter-flow elutriation protocol for the rapid and direct isolation of G1 cell cycle synchronised populations of both the procyclic and bloodstream form stages of Trypanosoma brucei that yields viable and proliferative cells. The high quality of the synchronisation achieved can be judged by the uniform DNA content, narrow size distribution, synchronous division, and the maintenance of synchronicity into subsequent cell cycles. We show that early-eluting fractions represent different G1 subpopulations that progress through the cell cycle with distinct temporal profiles post-elutriation, as exemplified by the observation of the maturation of a second flagellar basal body in late G1 phase, DNA replication in S phase, and dimethylation of histone H3 in mitosis/cytokinesis. We use our temporal observations to construct a revised model of the relative timing and duration of the nuclear and kinetoplast cell cycle that differs from the current model.