Hi there, when doing the intracellular cytokine staining for FACS analysis, take IL-17 for example, can the stimulate cells be fixed and permeabilized by Fix/Perm buffer in the presence of antibodies against IL-17A, means that staining at the same time? Is this a good strategy for better intracellular staining?
Waiting for your excellent suggestions!
Hi Tao Wu. In my opinion you can stimulate your cells and after you can perform the intracellular staining. A good strategy could be:
1 incubate your stimulated cells with surface antibodies that can help you in selection of your cellular population of interest
2 wash with PBS
3 incubate your sample with fixation solution
4 wash with PBS
5. gride the sample, add permeabilization solution and at the same time the fluorescent antibody for the intracellular stain (for example antibody anti IL-17)
If you suppose that in your medium there are other antibodies anti IL-17 they can compete with your fluorescent antibody and can prevent the binding.
Hello, Tao Wu!
I would also add that maybe you will get better results, if you use Fix/Perm buffer first and then add the antibodies in Perm buffer, as some fluorophores can be sensitive to compounds used for fixation.
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