I am a grad student & am having some problem in Gamma H2AX staining.
My interest is to see the effect of my gene on DNA damage after irradiation (5 Gy).
My problem is even my control cells (non-irradiated) showing many foci (some cells more than 10).
Here is my experimental setup
Cells: Mouse fibroblast cell line transfected with Human gene.
Untransfected cells grown in G418 & transfected cells with Hygromycin B.
- Cells were seeded in cover slip 24 hrs before irradiation (yes without antibiotic).
- Irradiated next day, incubated for 1hr.
- Then fixed and stained with primary antibody (Phospho-Histone H2A.X (Ser139), 1:1000) & then alexa fluor labeled secondary antibody ( 1:750).
Can anybody tell me how can I reduce the foci in control cells?
Thanks in advance