Antibodies against Phospho-Histone H3 mark the G2/M transition and stain the condensed chromatin just before chromosomal segregation. The nice thing about P-H3 serine 10 is that this is very specific and has a short half life of about 30-60 minutes.

Antibodies against Phospho-Histone H3 mark the G2/M transition and stain the condensed chromatin just before chromosomal segregation. The nice thing about P-H3 serine 10 is that this is very specific and has a short half life of about 30-60 minutes.

Cyclin A/CDK2 are highest at the G2 phase of the cell cycle whereas Cyclin B/CDK1 are highest at the M phase of the cell cycle. How will you detect these phases of the cell cycle?

Cells expressing phosphorylated Histone-3 at Serine-28 are in the M phase and thus this protein can be used as a marker to screen M-phase cells. So immuno- fluorescence with phospho Histone-3 antibody can help determining cells in G2 or M phase.

Thanks Prof. Dressler, Anne and Pritha for your helpful information.

The DNA content profile is also helpful: cells in G2/M phase generally have 4c compared to 2c of cells in G0/G1. If the DNA curve is bimodal this simple reasoning can be useful. This can be assessed with flow cytometry of cells stained with a DNA fluorochrome such as propidium iodide. Sure a second fluorochrome conjugated with an antibody marking some protein (i.e., those mentioned here before) could give more precise information.

Thank you Prof. Hoz. I was thinking to use PI with p-histone H3. But, in my microarray data, Cyclin A2, B1, E2, CDK1 and CDK2 are downregulated. Therefore, I was wondering whether the cells actually entered into M phase or stuck in G2.

You may also want to check if you have polyploid cells in your samples by cytometry: cells having 4N chromosomes can be diploid cells stuck in G2, or tetraploid cells in G1. You may even see some additional peaks (6N, 8N) if your cells accumulate significant rounds of endoreplication.

People often gate these cells out, and miss this information. Of course doublets still have to be gated out.

I agree with Vincent, polyploid and aneuploid cells must be taken into account particularly in cáncer populations. This makes more interesting the detection of some antigen phase-specific as Ruhul was searching. Cyclin B and CDK1 seemed good option but, if doubt about cells being in G2 or metaphase persists, microscopic observation could help since cells in metaphase can be clearly identified,

Thank you Prof. Coulon and Hoz for your thoughtful input. I have to check for polyploidy also.

There are some differences in phase specific expression between cells lines and cells derived from tissues especially with regard to cyclins. J.Pathol. 201:187-197 (2003). With the above in mind, your suggestions of Cyclin B1 and pH3 should be reasonable. Some of the G2/M checkpoint (CHK2 etc) markers can also be useful but again there are some differences between cell culture/cell lines and tissue derived cells. Geminin may also be useful in combination with pH3 to differentiate cells in prophase/metaphase with those in later stages of mitosis. Good reviews from Bell & Dutta and Ron Laskey's group in Cambridge. Hope this helps and I agree with suggestions above re ploidy.

Gregory, Pritha: what phospho histone H3 antibody are you using? I am trying to also sort out my population of 4N cells but the cyclin B1 expression is too high in all of the cells, I think I am going to give a try to the p'histone... any suggestion of your preferred antibody?

I am new on research gate, sorry if the conversation is out of date. However, as marker of the G2/M phase you can use some phospho-Cyclin B1 or phospho-cdk1/cdc2 antibodies. You can also try to separate nuclei from cytoplasms, Cyclin B1 accumulation in the nucleus is very high at G2/M, in particular the phosphorylated form (you can see it with IC as well). As control, you can also check the expression of Cyclins D, whose levels decrease at G2/M checkpoint.

To those who raised the ploidity issue: does the endoreplication (e.g. in Vincent's reply) occurs only in cancer cells? What drives endoreplication in these cells?

Thanks