Dear Wu, Any of the the dividing or activated or stimulated cell is likely to fall pray to effects of radiation and undergo apoptosis due to irreparable damage to DNA under the influence of master gene p53 and moreover, LPS may aggravate this damage due to enhanced free radical generation.

Hi, Naresh,

Thanks for your answer, but 25Gy is suppose to cause proliferation inhibition mainly according to some reports, will lower radiation does and LPS clear-out before radiation help in this case?

Considering the 25 Gy is such a large absorbed dose, you should consider doing a dose response curve using cell viability as an end point. Using a dye exclusion test would rapidly indicate the viability. I would suggest you wait one or two cell cycles before determining the viability.

Hi Alexa:

If radiation is not convenient in your experiment, you can also think about employing mitomycin C treatment (Current Protocols in Immunology).

However, if your aim is to use these DCs in MLRs, a colleague once told me that, if the percentage of CD11c+ cells is high (at least 90-95%), you may bypass this inhibition step (since the "contaminating" cells will be just a few, and won't modify your observations).

The MLRs I performed were evaluated by flow cytometry, so I measured specific LT CD4+ and LT CD8+ proliferation, and I did not pre-treat DCs neither with irradiation nor mitomycin C.

Good luck!