To characterize activation of immune cells, measurement of intracellular signaling using phospho-specific antibodies combined with immune phenotyping using surface markers is ideal. Owing to the unique capability of flow cytometry to measure events at a single-cell level, “Phospho Flow” allows for the identification of rare signaling events during processes such as co-stimulatory signaling and manipulation of checkpoint inhibition. However, combining extracellular and intracellular antibodies in a flow protocol can present technical challenges.
Topics in this presentation include:
- How small protocol changes can impact flow cytometry results;
- Examples of flow protocol optimization using phospho-specific antibody panels for T cell activation and innate immunity;
- Guidance on experimental design for combining phenotypic markers with phospho-specific intracellular targets;
- Discussion on the use of fix-and-perm kits.