DNA Damage Question
I am performing Phospho-yH2AX immunofluorescent staining of cells treated with either 0.1% DMSO or a drug inhibitor in order to measure DNA damage. Unexpectedly, I am observing high levels of signal in the DMSO treated cells. The cells are grown on Chambered Cell Culture Slides from CellTreat. It does not appear to be cell type specific given I have observed this with 5 breast cancer cell lines and 2 prostate cancer lines. I am using the CST Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718 antibody in Normal Donkey Serum at the recommend 1:400 dilution, so I don't believe its non-specific signal either. Has anyone encountered this issue before or know of any alternative explanations.
For reference I have attached an image below where H2AX signal is shown in pink these are completely untreated suggesting it is not an effect of DMSO
https://www.researchgate.net/post/Can_low_levels_of_ROS_induce_gamma-H2AX_without_causing_DNA_damage
https://www.researchgate.net/post/Gamma_H2AX_foci_seen_in_untreated_Hek293_cells
https://www.researchgate.net/post/What-do-gamma-h2AX-foci-represent
Perhaps the above RG discussions may be helpful.
Hi Dane,
Maybe you have already found the answer to your question, but what I read from your question can be answered if we take one aspect in consideration. Genomic Instability. Cancer cells are known to bear this as one of the causal features. With genomic instability, yH2AX occurance is normal. More the growth, more unstable the genome is. Before you come to a conclusion with this aspect, I would ask you to do a titration assay.
Grow the cells in 2.5% FBS, 5% FBS, 7.5% FBS and 10% FBS. Stain them. Lower FBS will lead to a slower growth. I expect you will see a gradient in the quantity of yHH2AX.
Somnath
Hi Dane,
I saw similar result across various cancer cell lines without any stress stimuli (better say without any specifically applied) and think, it indeed reflects basal level of genomic instability in such cells.
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