5 replies to this topic

[sen111]

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 with 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

[bob1]

Why are you using 2 different selection agents - to be scientifically valid you should be using the same on both transfected and untransfected.

If you are doing transient transfections, you shouldn't need to select for the plasmid.

Are you sure that G418 doesn't affect gamma H2AX?

Have you titrated the antibody or done peptide competition to confirm that the foci you see are due to specific staining?

[sen111]

Dear Bob1,
                   Thanks for your response.

That is because my cell line is a knockout cell line derived from a Knockout mouse. So control cells (Un transfected cell) need to be maintained in G418 (80ug/ml).
Where as transfected cell line (Stable cell line) is in Hygro B.

I also suspect antibiotic as a possible reason. may be i will try to culture the cells at least a week with out selection. Do you think will this help?

I have done antibody titration but not peptide competition.  I will look this peptide competition as well.

Cheers,
Sen111

[bob1]

So, if your stable KO cell line requires G418, that would mean that your stable transfection should also require G418 if they are derived from the KO line...  Have you checked to ensure that the stable transfected line is still KO?

I doubt that the KO mouse was constantly fed G418 to maintain the KO (I could be wrong though, so check this), if this is so, why do you need to maintain in G418?

[sen111]

Yes technically you are right. I need to add G418 even in transfected cells. But Stable clone express same gene, which was ko in mouse except it is of human origin.
So one express no gene (control) another express same but human gene (Stable). So even if KO lost it is ok.
Reason to choose mouse cell line is there is no human origin KO cell line is available. With these set up I want to measure Gamma H2AX foci staining.

Thanks again for your reply

[bob1]

OK, putting that stuff aside, did you titrate the antibody to ensure that you are getting specific staining?

I would expect some gamma H2AX foci in a proportion of a population of normal cells just from ordinary cells undergoing apoptosis (though I could easily be mis-remembering the little I knew about H2AX).

The drugs you are giving the cells don't induce H2AX foci?