The type of agent you choose determines what type of DNA damage you create and how these breaks will be repaired. Etoposide is a Topisomease II inhibitor and this creates DSBs with a protein (TopII) attached. This protein needs to be removed before the break can be repaired. With IR you generate clean DSBs and single strand breaks that are repaired mostly via NHEJ. 

Choosing the right agent thus depends on in which type of DNA repair you are interested in, or what type of aberrations you expect. Do you want to see if all cells form the foci, or do you wat to see if the breaks can be repaired and the foci disappear?

Dear Inger,

Thanks a lot for your answer. I understand it more now. 

Our candidate gene might be involved in NHEJ repair pathway (it's not very well characterized yet but was reported with some evidence). It seems I should go with IR option.

Regarding your last sentence,  I actually want to look at  both aspects: if all cells form the y-H2AX foci (to see if DSB can be formed-or with lower formation rate- in cells of our patient) and if the breaks can be repaired in this patient (based on the disappearance rate of the foci).

If so, is it still fine that I go with IR option?

Thanks for your help

One other thing that you may want to take into account is the time it takes to get approved for work on a cesium irradiator. At least here, it can take many months and background checks. So if someone in your lab is already approved or you can have someone else do the irradiation for you, you will be OK. If not, get started nowon the approval. Also, there are other agents to consider for dsbs as well. Bleomycin for one. Its an IR mimic for the most part

 Hi Nichole,

Thanks for the notice. Our facility uses  I never work with radiation before but had a chance to watch someone else use the machine (Faxitron X-ray irradiator in our facility) . I was told that I just need to register to use it. Will have to check it again then. 

Do you ever work with EBV-transformed cell lines? Most papers I read tested on EBV-transformed cell lines for optimization and use the optimized condition (drug concentration/radiation dose/time...) on primary cells. I don't know if EBV-transformed cells can make different effects on DSB assay (e.g due to EBV transformation) compared with primary cells? If primary cells are the best I need to arrange if I can estabish the assay at the time the patient gives blood.

Thanks. 

I think IR is a good choice then. Damaging the cells usually only takes a few minutes, so this method is also suitable to study repair kinetics. 

With the X-ray generator, you can irradiate X-rays, e.g., at 0.5 Gy/min. 1 Gy produces about 30-40 DSBs for which the number of gamma-H2AX foci reaches a maximum at 30 min post-irradiation. When you compare the repair capacity of different cell types (here, patient and non-patient cells), it will be intriguing/important to look at the time course, especially a later time, as the cellular response is affected by the residual DNA damage.

Article Histone H2AX Phosphorylation in Normal Human Cells Irradiate...

Please keep in mind that ionizing radiation induces DNA double strand breaks in addition to other various types of DNA damage, such as single strand breaks, crosslinks and base lesions.