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Question about isotype control in Phosphorylation staining

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Posts: 9

Joined: Tue Aug 07, 2018 3:09 am

Post Fri Aug 10, 2018 9:36 am

Question about isotype control in Phosphorylation staining

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.



Posts: 43

Joined: Fri Jul 10, 2015 1:04 am

Post Mon Aug 13, 2018 2:25 am

Re: Question about isotype control in Phosphorylation staini

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. https://lists.purdue.edu/pipermail/cyto ... 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.

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