Hi all,
I am doing intracellular staining of a group of cytokines namely IFNG, IL-17A and IL-4. I haven't stimulated my cells on healthy control PBMCs hoping to see these cytokines expressed/changed expression in patients. despite not using any activator or proton pump inhibitor I am seeing IL-17A stained in my healthy control, which encourage me to carry on.
knowing that, I have stimulated a sample of my cells separately (positive control) for each cytokine to make sure the antibodies working fine and they are, would anybody following the same protocol reassure me as all the protocols I have seen stimulate the cells in presence of proton pump inhibitor, but I choose to not manipulate my cells a lot!!
Thx
You can trust FCM data if you did proper optimization and if you have used all the necessary controls.
First, you should properly titrate every single antibody you use to minimize/eliminate aspecific signal of the negative population and maximize positive signal. This is especially important with intracellular stainings, as those have a much higher chance of aspecific binding to intracellular proteins. Ruud Hulspas wrote an excellent Current Protocols in 2010.
Then, to make sure you're looking at real specific binding, you should titer with unlabeled antibody.
Now come the controls. Ideally you need a negative and positive staining control. Positive is easy, just do what everyone does. Negative, mhm, you say you see staining in unstimulated cells...
As a gating control, if you properly titrated your antibodies, you can and should use an FMO. Never use an isotype stained sample as a gating control, that is not its purpose. There are other controls to keep in mind as well.
But before you dive in, you should ask yourself: what is the question I need answered? How will I get to my answer, and is it the best possible way to do it? How do others answer this kind of question?
If all of the above sounds like magic to you, please consider taking an advanced course in flow cytometry. Otherwise you'll be spending a lot of money and producing dubious results at best.
Hello Fatma,
I think constitutive expression of IL-17A is not unheard of, so might as well be the case for PBMC. I suppose, you have an unstained control that you run alongside your samples to test for a potential autofluorescence of your cells. As long as you see a change between unstained and healthy control that would be reliable data.
I hope this answers your question?
Thanks Svenja and Jonathan for ur helpful answers.
Just to clarify, I am running FMOs, SSC and unstained controls and gated IL-17 and IFNG population based on these controls specifically FMO, I am just concerned because I couldn't find a reference/paper looking at these cytokines without activation. If anyone please aware of paper looking at the intracellular cytokines without activation, would it be deeply appreciated to point it out.
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