Mastering Phospho-Flow: Optimizing Immune Cell Activation Assays with Intra- & Extracellular Flow Cytometry
Christopher Manning
Tuesday, May 22, 2018
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Chapters
Introduction
About Cell Signaling Technology
Topics
Why would I want to perform phospho-flow?
Phosphorylation is commonly detected by western blot, which has limitations
Live-cell flow cytometry is not compatible with immuno-labeling of intracellular proteins, including phosphorylation sites
“What protocol should I use for phospho-flow?”
A variety of fixation/permeabilization protocols will work for detection of phospho-proteins; start with a recommended protocol
Permeabilization reagents: the biggest difference among protocols
Your protocol options are dictated by the antibody and the target
Protocol flexibility exists for many antibodies, across multiple target types
“What protocol should I use for phospho-flow?”
“Why isn’t this antibody working in my protocol?”
Protocol changes may impact antibody specificity
Ensure your antibody is validated in the exact protocol you are using
Timing is critical for the detection of phospho-proteins
Fix/Perm kits are great options for antibodies validated with that kit
“Why isn’t this antibody working in my protocol?”
“How can I get my CD markers to work with phospho-Flow?”
Order of fixation and staining steps can be altered to improve performance
CD marker labeling may be impacted by reagents
Some fluorophores may be impacted by organic solvents
“How can I get my CD markers to work with phosho-flow?”
“Can you show me that it works?”
Six-color T cell activation assay (PBMCs +/- anti-CD3/CD28, 10 min)
Six-color T cell activation assay (PBMCs +/- anti-CD3/CD28)
You can do phospho-flow!
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