Flow Cytometry Guide: Signaling and Phosphoflow

Author: Mike Blundell | Reviewer: Chloe Fenton

What is phosphoflow in flow cytometry?
Phosphoflow (phospho-flow cytometry) is a technique used to measure intracellular signalling pathways by detecting phosphorylated proteins in single cells. It enables researchers to analyse kinase activity and cellular responses to stimulation at a specific time point using fluorescently labelled antibodies.

What is cell signalling analysis by flow cytometry?

In addition to the expression of surface molecules, there is a lot of interest in the activation state and measurement of signaling cascades. Flow cytometry offers a quick and effective way to measure signaling at a specific time point in individual cells.

The staining methods for detecting signaling molecules and phosphorylated proteins may differ from normal intracellular staining and specific experimental controls may be required. Typically cells need to be rapidly fixed to avoid dephosphorylation and stronger permeabilizaton methods may be required to ensure permeabilization of the nuclear membrane. As with cell cycle staining, fixation and permeabilization may alter your ability to measure cell surface molecules. Care should be taken with the buffers used as they can interfere with signaling e.g. EDTA.

Signaling can be measured using dyes which fluoresce in response to changes in calcium. Typically cells are loaded with dyes such as indo-1 to determine a baseline level of signaling and then stimulated. The change in fluorescence denotes a change in intracellular calcium levels.

How does phosphoflow work?

Typical phosphoflow workflow:

1. Stimulate cells (e.g. cytokines or ligands)
2. Rapid fixation to preserve phosphorylation state
3. Permeabilisation to allow intracellular staining
4. Staining with phospho-specific antibodies
5. Flow cytometry acquisition and analysis

Which method should you use to measure cell signalling?