Problem with Gamma H2AX foci staining

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sen111
sen111's picture
Problem with Gamma H2AX foci staining

 Hello all,
                    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
Cheers
Sen 

glykys
glykys's picture
Dear Sen,

Dear Sen,
I have just started to learn this tecnique and really I am not experienced about this. But the resarcher whom is experienced and try to teach me the important points of this issue said that first of all you must use fresh cells. For your cell cultures if you are passaging your cells more and more times then you can see many foci in the control cells. so if your problem is just about control cells, I think this is not a problem about the experimental procedure, it may be only about cell culture period. Try to use fresh cells. Good luck.

Gökçe 

Pharmaseed
Pharmaseed's picture
Hi, Try to lower your

Hi, Try to lower your irradiation dose to 2Gy and also i propose to expose the cells to gamma IR on 24 wells with medium and only after that to fix them and transfer to slides (maybe the fixation permabilizes your nuclei). Also try to lower yor antibody concetratios.
Good luck