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Question about isotype control in Phosphorylation staining

PostPosted: Fri Aug 10, 2018 9:36 am
Hi everyone,
I'm new here and I have used CyTOF for 2 years, but I still have a question that I don't know how to explain.
When I do my experiment about Phosphorylation, I have never found anyone who use Isotype control, why? Isn't it necessary?
Thank you.

Re: Question about isotype control in Phosphorylation staini

PostPosted: Mon Aug 13, 2018 2:25 am
by BjornZ
Isotype controls should only be used to look for non-specific binding, and not as the basis for setting a "positive" gate as you need in phospho-flow. Mario Roederer and others have explained why a few times on the Cytometry list, e.g. ... 09860.html.

CST (who makes the majority of the phoshpo antibodies we use) does an excellent job validating their antibodies, so we've deferred to them for matters like non-specific binding. That leaves no real use for isotype controls in phospho-flow.

For setting a "positive" gate, it's best to look at an unstimulated condition in the same cell type, vs. a stimulated condition, or just look at median intensities for more subtle shifts.