Mastering Phospho Flow: Optimizing Immune Cell Activation Assays

Combined analysis of immune phenotyping and rare signaling events can be performed using flow cytometry, but presents technical challenges. Topics covered in this workshop include:
•Guidance on combining surface markers with phospho-specific intracellular targets;
•How small protocol changes can impact flow cytometry results;
•Examples of optimization using antibody panels for T cell activation and innate immunity;
•Discussion on use of fix-and-perm kits

Presented by Chris Manning, Group Leader of Flow Cytometry at Cell Signaling Technology.

When you think about what you're usually doing by live-cell flow, you've got access to 370 CD markers and about 1400 proteins on the plasma membrane surface. Of course, if you fix and permeabilize those cells, now you're talking about an order of magnitude more proteins you can detect and over 400,000 modifications on those proteins as measured by mass spectrometry. That's really where the biology of these cells is driven. These are the readouts that let you understand what's going on in the cell. What is the cell doing? Where is the cell headed in terms of differentiation or activity?

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