Abstract
Fibroblast cycle synchronization in G0/G1 is an essential step for nuclear reprogramming by cloning or induced cells to pluripotency. Considering the diversity among rodents and the ecological and scientific importance of these animals, we compared the contact inhibition, serum starvation, and 10 µM of roscovitine as methods of synchronization of red-rumped agouti fibroblasts. The effects of each protocol were evaluated on the percentage of cycle phase, morphology, viability, and apoptosis levels. The results showed that culturing the cells to serum starvation for 24 h (75.9%), 48 h (81.6%), 72 h (86.2%), 96 h (84.0%), and 120 h (83.7%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (31.4%). Also, this effect was not different between the times of 48 and 120 h (P > 0.05). A similar response was observed for cells cultured with roscovitine for 12 h (86.9%), 24 h (74.8%), and 48 h (81.7%), with a higher percentage of synchronized cells in G0/G1 compared to cells not submitted to any synchronization treatment (52.2%). Nevertheless, this effect was best evidenced at 12 h (P < 0.05). Also, the contact inhibition for 24–120 h could not synchronize cells in G0/G1, with values ranging from 70.9 to 77.9% (P > 0.05). Moreover, no difference was observed for morphology, viability, and apoptosis levels in any synchronization method (P > 0.05). Therefore, serum starvation is as efficient as roscovitine on cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.
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Acknowledgements
The authors thank the CEMAS/UFERSA for providing biological samples from the red-rumped agoutis and to the Laboratory of Experimental Oncology for their assistance in the use of the flow cytometer.
Funding
This study was financed by the Brazilian Council of Scientific Development (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES, Financial Code 001). C Pessoa, MF Oliveira, and AF Pereira are recipients of CNPq grants.
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Praxedes, É.A., Oliveira, L.R.M., da Silva Viana, J.V. et al. Serum starvation is as efficient as roscovitine on the cycle synchronization in G0/G1 of red-rumped agouti fibroblasts. In Vitro Cell.Dev.Biol.-Animal 60, 249–257 (2024). https://doi.org/10.1007/s11626-024-00866-7
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DOI: https://doi.org/10.1007/s11626-024-00866-7