In this post, I discussed the importance of testing and titrating the stimuli used for intracellular cytokine staining. Today let's look quickly at another critical aspect: fixation.
Fixatives crosslink proteins on and in your cells. This allows us to then punch holes in the membrane using detergents, removing the lipids and allowing antibodies access to the interior of the cell. If don't first fix, a lot of cells will break apart in the detergent permeabilization step.
Cytokines are about ten times smaller than an antibody. So, any hole an antibody can get in, the cytokine can probably get out. The fixative, then, probably crosslinks cytokines to other proteins nearby, creating multi-molecular complexes that are much bigger. If we don't fix adequately, or we try to create membrane holes while fixing (fix/perm), we're much more likely to have cytokines leak out of the cell, reducing our ability to detect them with intracellular staining.
IL-2 staining in mouse and human CD3+ T cells following fixation with different buffers:
The cells here have all been stimulated for four hours with PdBU and ionomycin, using brefeldin A to block secretion. Staining is overnight.
The buffers designed to allow access to the nucleus (True-Nuclear and Foxp3/Transcription Factor) abrogate murine IL-2 staining as if the cells hadn't been fixed at all. With human cells, those buffers reduce the intensity of the IL-2 staining 3- and 10-fold, respectively.
Not all cytokines behave the same way. IL-2 and IL-4 are particularly sensitive to fixation conditions and can be completely lost. TNF has almost no sensitivity, perhaps because TNF is first expressed as a transmembrane protein rather than in secretory granules. Most are somewhere in the middle.
IL-4 and TNF staining in mouse CD4+ T cells:
Good fixative options for cytokine staining in no particular order:
4% PFA
Formalin
Thermo IC Fixation buffer (kit version of 4% PFA)
BioLegend Fixation Buffer (kit version of 4% PFA)
BD CytoFix (kit version of 4% PFA)
What is the difference between PFA and formaldehyde? I thought they were the same, but one powder, the other soluble. And can PFA permeabilize directly, without saponin? It seems to from the text, but I couldn't settle. Would you then use perm/buffer from Foxp3 kit for the staining after 2-4% PFA fix/perm?