Abstract:
Objective: Cell cycle for somatic cells is composed of interphase including G1, S and G2, and the M phase. In vitro cultured cells usually exist at various cell cycle phases even though they originated from one mother cell. To analyze specific variations of some proteins, genes or other cellular components it is essential to keep the cells at or bring them to the same cell cycle phase at the maximum level; this is called cell synchronization. Thus, we analyzed the use of benzimidazole derivative nocodazole, a microtubular depolymerizating agent, for cell synchronization. Material and Methods: To synchronize in vitro cultured Vero cells 0.3 mu M nocodazole was used. When untreated cells were considered the control group, the experiment group consisted of cells that were treated with nocodazole for 6, 12 or 18 hours. In the recovery group, following nocodazole treatment, cells were washed and cultured in nocodazole-free medium for 6, 12, 24 or 48 hours. After culture periods, cells were fixed and microtubules (after anti alpha+beta tubulin antibody, FITC) and nuclei (7-AAD) were labeled with fluorescent material. Slides were observed with a confocal microscope and the results were statistically analyzed. Results: Nocodazole kept the cells at mitosis, time dependently. Disrupted microtubules were seen in the cells particularly during mitosis. When nocodazole was removed from the culture medium, cells completed their mitosis and resumed to the interphase stage. At 12 hours of recovery, almost all cells (99%) were in the interphase. This result indicates that nocodazole synchronizes Vero cells. Besides, we also observed that nocodazole induced apoptosis in Vero cells. Conclusion: We noted that nocodazole administration at the appropriate time and dose might be used successfully to synchronize the cells in culture.
