I have been using two different protocols for cytokine intracellular staining and getting different results. I used eBioscience and BD Bioscience.
The first protocol did extracellular staining for surface markers, Perm/Fix'ed the cells, washed with Perm buffer, Fc blocked (?) cells with blocking buffer, incubated with specific/-ve control Abs, washed again with Perm buffer and proceeded to FC analysis.
The second protocol did Fc blocking before application of the extracellular Abs, washed cells, Fix/perm'ed cells, washed with Perm buffer, stained cells intracellularly with specific/-ve control Abs, washed with Perm buffer and proceeded to FC analysis.
I admit that neither can I understand the point of an intracellular Fc block step, nor have I ever read or come across such a prodedure. Can anyone propose why such step should take place and whether it is safe for the insider of the cells and for the reproductivity of the experiments (strangely enough, I 've had more background with the second than the first protocol).
the purpose of the Fc block is to bind up the FcR on the cell surface. That is usually the first step. I bet your first protocol yielded higher numbers of positive cells. The second protocol is the BD protocol which is pretty standard. Ignore the first protocol. Go with the second one.
I've never stained for cytokines, but the second protocol makes absolutely no biochemical sense to me. When you fix your cells, the PFA/GA reacts with the amino groups on the protein and sometimes it reacts completely, but sometimes it leaves open unreacted aldehyde groups. If you add any protein to it - it's going to bind non-specifically to these reactive groups, which is why you're getting more background with the second protocol in the -ve control.
I always pre-block with serum and then apply my antibodies in serum. If serum is not an option, you can try just amino acids (e.g. 100mM glycine? just don't forget to pH it.
Also, I always fix, then permeabilise and then block. Since PFA/GA can get inside the cell (and permeabilises it to a little extent), but the proteins in the serum can't.
Hi I have done many intracellular stainings in T cells wich I have cultured (RPMI1640 + 10%FCS) before staining. T cells don't require FC blocking but if you stain cytokines in Monocytesn or granulocytes the second protocol is standart, I aggree with Peter. Do your FC blocking then the surface markers and than perm/fix followed by the inkulabtion of your cytokine AB in permbuffer. Good luck with your staining!
Thanks for the support Claudia. Since we stained peripheral blood mononuclear cells, or whole splenocytes, or whole lymph node cells (T, B, mono, dc, etc), Fc block was standard.
Some amount of FcR can be kept inside the cell and this is why when you first block (on the surface) and then permeabilize/fix, you may still have a background: because of binding of specific antibody with intracellular FcR that are not blocked.
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