isotype-control in FACS shifts heavily - (Sep/19/2005 )
For my research project, I radiate glial cells and measure levels of Apo-1 (CD-95).
When I do so, I see a nice increase in CD95-receptor and -ligand-levels in a time- and dose-dependent manner. But my isotope control both for receptor and ligand shifts almost accordingly, so now I wonder whether the effect I see is completely unspecific (Even though I have no clue why) or if there could be something wrong with my isotope control.
What possible error sources you know I could check?
I use a jo2-antibody (FITC-conjugate) and mfl3-antibody (PE) from BD, my control are FITC- and PE-labeled Hamster Ig's, respectively.
It's possible your cells have high levels of Fc receptor, which are binding your isotype and test antibodies. Try blocking your cells with 10% serum (or Fc fragments/anti-FcR abs but serum is easiest), then staining with your abs. Hopefully this should eliminate any effect of Fc receptors and you should see low background with the isotype ab. If you still see a shift with your specific ab you know it's the real thing.
All the best,
first of all, thanks for your fast response, I will try it tomorrow and hope it works.
Still, I'm a little sceptic, as I can't imagine why the isotype would then shift accordingly to my standard?
If irradiating your cells also up-regulates Fc receptors aswell as Fas and FasL, that could explain it. (I've never worked with glial cell but I know certain cell types e.g. macrophages express alot of Fc receptors). If this is the case blocking should reduce the non-specific background with your isotype and hopefully your specific anti-Fas/FasL staining should be visible. I know someone who may have experience with glial cells, if you're still having problems I could ask him for advice.
By the way let me know if it works. I guess I could be wrong.
I have prepared my cells today, I'll radiate them tomorrow and measure on friday, then I'll let you know. But thanks first of all for your response!
Did it work? I am having a similar problem with isotype signals shifting where the specific antibody is binding. We were only using PBS because FBS interfered with our Ab-biotin, Streptavidin-fluorophore. We are going to add BSA to alleviate this problem but I thought I would see if it worked for you. Let me know!
Sorry for not answering sooner.
I redid the experiment twice, and prepared Realtime-PCRs.
All efforts were negative (isotype still shifted, and RNA didn't increase), so I suppose that the increase is unspecific.
I noticed though, that mycells increased in size upon radiation, so I think that might be the reason for the higher rate of unspecific binding. (Tried only FBS to block, though)