I need help with a cell cycle synchronisation protocol for MCF-7 cells. I have tried the following methods: 1) 0% serum starvation for 24 hours, b) 0% serum starvation for 32 hours, c) 0% serum starvation for 72 hours, and d) 1% serum starvation for 72 hours.
Each method has some varying degree of success; however, the longer the cells are starved, the more sensitised they become to subsequent treatment. Any addition of compounds such as methotrexate or curcumin, which are S phase and G2M phase blocks, result in cell death. I have lowered the concentrations of the compounds but it still does not work.
Unfortunately, we do not have thymidine, lovastatin or hydroxyurea, and therefore, need help with alternative protocols. I am aware of centrifugal elutriation and mitotic shake-off techniques but I am unfamiliar with the the methodology.
Thanks!
Hello Nomfundo Nzama
Please refer to the research article below for various methods for synchronizing cells which also includes mitotic shake-off technique. It may be helpful to you.
Article Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
Best Wishes.
Dear Nomfundo Nzama,
The absence of serum in the medium is not a synchronization treatment in itself, but can be used as pre-synchronization in combination with other treatments. To get good results, you need to invest a little in this. Thymidine and hydroxyurea are inexpensive and readily available reagents. You can read about the different methods for cell synchronization in my special review:
https://www.researchgate.net/publication/297946278_Review_Analysis_of_the_cell_cycle_and_a_method_employing_synchronized_cells_for_study_of_protein_expression_at_various_stages_of_the_cell_cycle
Best wishes,
Rustem Uzbekov
Is it a new scientific question that different starvation time determine cell cycle distribution?
Dear Nomfundo Nzama!
You also look at the following protocol of synchronization of MCF-7 cells
Article Simultaneous Inhibition of Cell-Cycle, Proliferation, Surviv...
Cell cycle analysis
MCF7 and MDA-MB-231 cells were plated in 60 mm dishes (1x106cells/dish) and were allowed to attach for 24h. The cells were synchronized by culturing them overnight in media with 1% FBS. The cells were washed twice with PBS and the culturing media was replaced with fresh media enriched with 10% FBS along with the super combination of the six phytochemicals. At time points of 0, 6,12, 24 h the cells were harvested by trypsinization, washed with PBS, fixed overnight in 70% ice cold ethanol, and the DNA was stained with propidium iodide after RNAse treatment according to standard protocol [27,]. Cell cycle analysis was performed by flow-cytometry (BD Biosciences, USA) and cells in G0/G1, S, G2/M and the sub-G0/G1 (apoptotic) phases were quantified.
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