I have performed FACS for a protein by intracellular staining using the antibody specific to my protein. I have kept all the possible controls (unstained, isotype control and also primary and secondary antibody control for untreated) for the FACS.
Below I have attached an image. I see that upon treatment (lower panel) there is positive staining as seen by shift of cell population along the APC axis. Upper panel is without treatment. The SSC and FSC for untreated and treated show similar scatter of cells, so there is no change in morphology of cells.
The question raised by my reviewer is "Intracellular labeling to detect the protein positive cells looks weird as the whole population shifts instead of a sub-population staining clearly positive."
Is it really weird?
Can anyone help me with this? What is the possible explaination for this shift and what can be done to rectify it?
Thanks in advance
It's hard to say if it looks weird w/o knowing the marker you are staining or the treatment. My guess is, yes. Also, I think the way you have presented your FACS data is confusing. I'm also not sure why you have the intracellular stain plotted vs. SSC. Is this part of your study? Do you expect a difference in intracellular marker as cells become more granular? If not, then lose that as your y-axis. I will often place my IC stain on y-axis and FSC on x-axis, since lymphocytic populations have a typical size pattern with light scatter.
@paula - Thanks.
The marker is a viral protein. the treatment is viral infection. I want to quantify the number of cells infected with virus in the whole cell population. I am not expecting any difference in staining due to granularity of cells. I followed the suggestions you gave but still there is shift in the whole population as i saw with IC vs SSC
Ok. So, that leads to the question; is it possible that ALL your cells are infected? Because that is what it looks like. It could also be an artifact of fixing/permeabilization/staining process. In addition to an isotype control (which you really need to include on your figure) you probably want to have a control stain or a "competed" stain. Maybe antibody against a different virus??? Or you could show specificity for the stain by using 5-10X concentration of non-fluorochrome labeled antibody before adding the fluorochrome-labeled (APC) antibody. If this take the staining back to isotype or uninfected control levels, then it is a specific staining. OR, if that's not doable for what ever reason, you could do a titration of virus infection, showing a dose-dependent increase in percent positive with the addition of the virus to culture (I am assuming in vitro infection).
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