Cell cycle analysis. - Cell synchronized or no-synchronized. (Jul/09/2008 )

Hi everybody,

I'm performing cell cycle analysis by propidium iodide staining and flow cytometer analysis. I want to analyze the influence of an agent in the cell cycle phase in my cells. I don't know if I have to synchronize my cells or not. What is the purpose to synchronize or no-synchronize? In which cases you need to syncronize them? What are the differences of the results in both cases?

Thank you

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At least as controls, you will want to have chemicals that can cause cell cycle arrest in different phases. I used to use Nocodazole to arrest in M (4N DNA peak) and aphidicolin to arrest in S phase. Aphidicolin treatment is also nice for synchronization as cells generally resume S phase rapidly/synchronously after removal of the drug. (for mammalian cells)

For your purposes, you will probably first want to see if it causes cell cycle arrest on its own, so you will not need to start with synchronized cells. To more specifically define how your agent causes cell cycle arrest (if it does), you may want to start with a synch'ed population.

One reason this might help you is to see at what point in the cell cycle your agent acts. The resultant cell cycle arrest morphology following agent-treatment does not necessarily indicate the point in the cell cyle your agent acts. For many agents (such as aphidicolin and nocodazole) the arrest-phase is the same as the phase where the agents act. Other drugs may inhibit upstream regulators that are essential for progression through the proceeding cell cycle phase. So if you expose cells to the drug after the regulator has acted, the cells will complete another cell cycle before arresting.

hope this helps
Dan

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I totally agree with Dan. Nocodazole will arrest in G2/M phase (if memory serves) and can be easily removed from mammalian cells following a wash and addition of fresh media.
It would be a good idea to sync your cells as controls.

Happy hunting!

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Thank you very much.

I'm new in this field and sometimes, I'm losing.
So, you recommend synchronized cells as a positive control? What things I could see with synchronized cells that with non-syncronized cells I couldn't?

I've analyzed my first experiment. There are a increased population in G2-M phase. If this result is confirmed with more experiments, the next step is to synchronize the cells? With which purpose?

Thanks a lot!!!!

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Besides helping verify your FACS histograms, the positive controls can tell you are the percentage of cells that are cycling. Normally HeLa and healthy tumor cell lines will all be cycling in pre-confluence growth conditions, but it should be checked. If after 18-24hr, nocodazole causes complete 4N arrest, but your drug doesn't, you can at least say your drug is less efficient at G2/M arrest (or perhaps your drug is affecting the progression at another point in the cycle as well). However, if after Nocodazole treatment you still see 20% of the cells sitting at G1, you will know that you can probably only hope to get 80% arrest with your drug. When you expand your studies to include various cell types, these controls will be especially important.

And as I said originally, synchronization will help you define when in the cell cycle your drug acts. You can get creative and test various mitotic kinase inhibitors, tubulin stabilizers&destabilizers, as well as replication inhibitors. And proceed to co-sensitivity studies with cancer compounds...
Sounds fun.
Dan

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Thank you very much!!!
It is so helpfully.

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Dear All,

I found out that here there are some experienced people with cell cycle arrest. I am going to arrests my cells which are African green monkey kidney cell line in G2/M using nocodazole. I have to optimize the concentration and time of application of this chemical. I was wondering if anyone has any ideas or references about that. I do appreciate your help.

THNX

Haki

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