We are using gammaH2Ax as a biomarker for DNA damage in HeLa cells after exposim them to different doses of ionizing radiation.
Samples at 0 and 0.5 h from irradiation look normal, however, samples at 24 h from irradiation (both 0 and 4 Gy) display a strong signal from the whole nuclei for the filter corresponding to gammaH2Ax.
Anyone had similar experience or any idea about what could be wrong?
The protocol works for other cell lines- glioblastoma, fibroblasts.
And we had very high reproducibility (4 experiements in total).
Hi Roxana,
strong gH2A.X signal after 24h indicates that the DNA is severely damaged. After low irradiation dose and/or short incubation time after exposure you normally see only some small dot-like gH2A.X foci in the nucleus, but with the high dose you can get thousands of them so that the nucleus appeares to be stained homogenously. The only thing that shouldn't happen is the positive staining of 0 Gy samples after 24h. But since it is reproducible you should take it as it is, namely as a "biomarker for DNA damage" indicating that even unirradiated cells somehow experience DNA damage during your experiment, may be indirect via other stress stimuli (temperature or pH shock). Other cell lines might be less susceptible for such perturbations explaining why your protocol works for them.
I did previously some gamma-irradiations on HepG2 cells to analyze gH2A.X signal, and since we didn't have a X-ray source in our institute I had to go with my cells to a neighbouring building. Interestingly, the way alone was sufficient to give some DNA damage signal as confirmed by an additional 0 Gy sample left in my institute.
Please excuse my questions to understand your question!
[1] Do you mean that at 0.5 h, does each nucleus of 4 Gy irradiated cells bear many foci (ca 120-160), but a few or less in 0 Gy (sham) irradiated cells?
[2] Do you mean that at 24 h, each nucleus of 4 Gy irradiated cells and 0 Gy (sham) irradiated cells both bears many foci? Does the whole nucleus or whole cell have fluorescence? Do you see fluorescence with other filters? Auto-fluorescence?
[3] Cells in S-phase can be positive with gammaH2AX independent of the treatment to induce DNA double strand breaks, but how is the cell cycle distribution?
The following papers report the data on gammaH2AX foci in HeLa cells at 24 h after exposure to 4 Gy of X-rays (and energetic carbon ions as well in the first paper).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794881/
http://cancerres.aacrjournals.org/content/70/7/2984.long
Apoptotic cells are generally have more foci, which may be the case at 24hr. For maximum number of foci wait for more than 0.5hr. microscopic setting may be revisited!
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