Discussion

Centrifugal counter-flow elutriation is a superior, non-invasive method to synchronise cell populations. The centrifugal force within the centrifuge is counterbalanced by a simultaneous flow of buffer that is being pumped through a special elutriation chamber in the opposite direction. Particles are thus separated by size, with smaller ones eluting first at low buffer flow rates if the centrifuge speed is kept constant. Since Trypanosoma brucei (procyclic form) PCF cells increase by several microns in length during the transition from G1 to S phase of the cell cycle, the isolation of extremely pure (>95%) G1 fractions by elutriation is possible. These cells can then be put back into culture and will synchronously proceed through the cell cycle. Bloodstream form (BSF) cells are not only smaller than PCF but also highly motile, and have thus far not been reported to separate efficiently during elutriation. Here we present data showing that isolation of cell cycle phase enriched populations of BSF cells by elutriation is possible and more reproducible than hydroxyurea-mediated synchronisation. However, greater care has to be taken that the starting population is in the logarithmic phase of growth to ensure subsequent synchronous progression through the cell cycle. Our overall goal is to use these optimised elutriation procedures to produce cell cycle stage enriched samples from both BSF and PCF cultures for comparative proteomic and phosphoproteomic analysis to identify novel cell cycle regulators in the parasite.