Cell cycle synchronisation

HCT116 cells were seeded in 12-well dishes and treated with 20 μM Lovastatin for 24 hr to induce G1 arrest (Rao et al., 1999). Cells were washed and medium containing 2 mM Mevalonic acid (MVA) added to release the cells from the arrest. 8 hr after release, 9 μM RO-3306 was added to hold cells at the G2/M boundary (Javanmoghadam-Kamrani and Keyomarsi, 2008; Vassilev et al., 2006). After 28 hr in RO-3306, cells were washed and drug-free medium added allowing cells to enter mitosis. Flow cytometry was used to analyse synchronisation efficiency, and to establish and optimise the procedure for the experiment in Figure 2 based on assessment of cell cycle progression kinetics.