Hi Marina,

Do you need to fix your cells before doing FACS? If your antibody is suitable for live staining of cells I would not fix them. Did you know that PFA fixation can also "open" the cells? I was doing live cell staining of my protein of interest, which is evenly distributed ober teh plasma membrane (and hence nicely delineates it), and then fixed the cells for further immunochemical analysis. Having a look at the cells was really impressive as one could see holes in the plasma membrane. Hence, your antibody might penetrate the cytoplasm and increase signal intensity (either by binding to intracellular GPCRs or by unspecifically binding to some other protein). Hence, PFA fixation (even without permeabilization with Triton etc) is not a reliable procedure to detect cell surface antigens. Live cell staining is better suited but should be rather short (5-10 min max) to avoid antibody-induced internalization of your protein of interest.

Hope this helps.

Best,

Jochen

have you done something like a negative control. Fixed cells without any antibody-treatment? You can compare your results and find out, if it is an artificial signal.

Thank you Gudrun,

Of course we always do all necessary controls, including cells only (no antibodies) and isotype-matched control and it is all negative. However I have heard (without specific proof though) that some antigens are particularly prone to non-specific staining and this is making me worried that our antigene is one of those because we see very little staining on unfixed cells. I myself however can not come up with any logical explanation as to why fixation will cause any non-specific binding of antibodies.

You have done the right negative control. An explanation could be that the Ab is generated against an epitope that is obscured in the protein native state. After the fixation the epitope may have been exposed. The Ab is maybe generated using a linear artificial polypeptide. If the epitope is in the normal protein inside a globulin like structure is it shed from the Ab. Or the Ab hase been raised agains a PFA-fixated protein or polypeptide. The epitope of the resulting Ab may be a cross linked structure that is only present in the cells after fixation. Helpfull?

Very helpfull, thank you. I thought so myself that fixation may stabilize/expose the epitope that ABs recognise. And this is potentially more important for GPCRs, which we are working with. But I'm also wondering if the ABs can bind a more generic "structure" rather than a particular aa sequence (epitope) and that is what we are seeing here. I also will be very interested to know if anyone else has come across similar observation with fixed/live cells?

PFA is a cross linking fixative and so can definitely change the antibody/antigen affinity based on the change in protein structure that occurs due to this. It's a common problem in IHC.

The only way to test it, I guess, is to over express your protein in a cell line and use an untransfected control cell and then test your assay with fixed and unfixed cells.

Fixation definitely changes protein conformation, thus affects downstream staining.

I have opposite experience. I work on one cell surface marker that loses signal after fixation. What I do is to stain the cell surface marker first and then do fixation for next step intracellular staining. In that way, the cell surface protein signal won't change. 

I have come across the opposite effect of loss of staining due to fixation as mentioned before. Maybe you could check your antibody on a different cell line where you get a somewhat better staining before fixation and than test if that increases as well with fixation? Another test might be using a different fixation method, e.g. Methanol instead of PFA or try changing the PFA concentration.

Hi Marina,

Do you need to fix your cells before doing FACS? If your antibody is suitable for live staining of cells I would not fix them. Did you know that PFA fixation can also "open" the cells? I was doing live cell staining of my protein of interest, which is evenly distributed ober teh plasma membrane (and hence nicely delineates it), and then fixed the cells for further immunochemical analysis. Having a look at the cells was really impressive as one could see holes in the plasma membrane. Hence, your antibody might penetrate the cytoplasm and increase signal intensity (either by binding to intracellular GPCRs or by unspecifically binding to some other protein). Hence, PFA fixation (even without permeabilization with Triton etc) is not a reliable procedure to detect cell surface antigens. Live cell staining is better suited but should be rather short (5-10 min max) to avoid antibody-induced internalization of your protein of interest.

Hope this helps.

Best,

Jochen

Thank you very much everyody for your valuable suggestions/comments. We have tried to investigate this problem in a bit more detail. This phenomenon of significant increase in staining after fixation is definetely cell type specific, but not cell line specific, and we also observed it with more than one receptor. I like the suggestion of Jochen that  PFA is permeabilising the cells so we will try other fixatives and also try to play with PFA  concentration.

If antibodies works on fixed cells or not varies from clone to clone. Most antibodies for FACS have been selected on unfixed targets, so it is "by chance" whether they also stain fixed or not. I have experienced all three cases; works as well on fixed as unfixed, no staining on fixed, or unspecific staining on fixed cells. In our hands, it is often the monocyte population in PBMC that stains unspecifically. it could eg be like someone here suggests, that some cells are "sticky" to antibodies on the inside, disrupting the membrane might give this effect. If you can use other markers to identify the cell population that you expect to be positive, it could help. Otherwise, try to find another antibody clone for the same antigen, or if possible stain unfixed cells and fix afterwards, which is the routine procedure for FACS.

Just to add to all the wonderful answers here already - I have been doing lots of phosphorylation assays using Flow cytometry  and also Intracellular cytokine assays mainly in the context of T cells - we had similar problems loss of signal after PFA fixation - for a antibody selection guide there is this website http://cytobank.org/facselect/ where people have already tested different antibodies and how the fixation affects them - you can check it out and see if your antibody clones are resistant to harsh/mild fixation. 

Aldehyde containing fixatives (including PFA) are known to cause fixative-induced fluorescence. Its worth trying an aldehyde independent way of fixing the cells. http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf