Hi everyone!
I'm trying to measure the cell cycle progression of HeLa cells, to complement other measurements involving cell grown. I know that the most used method is flow citometry, determining the DNA content by the use of i.e. Propidium Iodide. However as the DNA content is the same in G2 and M, I can't diferenciate between these phases.
So, there is another marker that you could suggest? and also do you have information about the kinetic of the HeLa's cell cycle, like how many hours do each phase lasts post starvation/confluence/another type of syncronization/etc?
Thank you very much!
With HeLas, you can synchronize using a double thymidine block which will synchronize them at late G1/early S phase. After washout, about 3 hours after release they should enter G2. You can use detection of Cyclin A/CDK2 and Cyclin B/CDK1 as well as phosphorylated Histone H3 to detect G2 or M phase.
An advanced method is the FUCCI system. It's a multi-color reporter which shows a different color depending on the status of the cell cycle. It's definitely not high throughput but gives you detailed information about the status on the single cell level. With this method you can also address every kind of question concerning duration etc. by analyzing with long-term imaging microscopy.
Hi Aldo,
I would also recommend the FUCCI Cell Cycle Sensor.
But you can also try a Phospho-Histone H3 (Ser10) Antibody to identify mitotic cells. We combine this with propidium iodide for cell cycle and Click-iT EdU to identify S-phase cells.
Thank you very much Anthony, Rainer and Chris for your advices!
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