We need to investigate cell cycle distribution after irradiation. The commonly used antiobodies, for example, pHH3 is not recommend since it will be dephosphorylated during G2-M cell cycle arrest after irradiation, which may result in wrong data interpretation using FACS.
To keep into consideration is not only the fact that H3pS10 becomes dephosphorylated after damage but also that you lose your mitotic population upon irradiation. Cells that have already entered mitosis will continue through and out of mitosis and those irradiated before the G2/M boundary will be prevented from entering mitosis by the checkpoint. In our work, we circumvented this by harvesting 30 minutes after irradiation, which retains cells that had already entered mitosis at the time of irradiation while they progress through (mitosis lasts about an hour). You can see an example of this by Flow after 2h Phleo in Fig 2B Giunta et al 2010 JCB, where the mitotic population is reduced to 0.4% (this is similar after IR). So, it's not only the mitotic market to pay attention to but also you experimental timings to be sure that you still have mitotic cells in your irradiated sample.
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