Signaling pathways

Phosphospecific flow cytometry

Signal transduction is a process of communication within the cell that consists of controlled cascades of protein modification. It can occur as a result of direct interaction between neighboring cells or detection of cell-signaling molecules, which include nutrients, oxygen, growth factors, and hormones. Complex networks of multiple signaling pathways allow cellular responses to be coordinated and highly sensitive. Dysregulation of these pathways can lead to various disease states, that’s why signaling molecules often become the aim of scientific investigation leading to development of effective therapies.

The membrane receptors are starting points of many signaling pathways. Such receptors are classified into several groups, including enzyme-linked receptors, G-protein-coupled receptors (GPCRs), and ion channel receptors. For example, the receptor tyrosine kinases (RTKs) belong to the first group and exert physiological effects via protein phosphorylation and dephosphorylation events. Being implicated in many pathologies (cancer, atherosclerosis, and diabetes) enzyme-linked receptors are an attractive therapeutic target.

Phosphospecific flow cytometry is uniquely suited to study intracellular signaling pathways, because cell types can be distinguished by surface markers and simultaneously analyzed for phosphorylation states of signaling proteins. In phosphoflow, cell populations are stimulated with a ligand specific to a membrane receptor. Then, the cells are fixed using paraformaldehyde, such treatment preserves the state of proteins within the cell. In the next step, detergents permeabilize the cell membrane, allowing fluorescently labeled antibodies to enter the cell and bind to the phosphorylated proteins.

Phosphospecific flow cytometry can complement analysis of cytokine receptor expression, as it can be correlated with testing of responsiveness to their cognate cytokines and show actual functional activity in addition to surface expression.

Phosphoproteins extensively studied by flow cytometry:

Table from Sara de Biasi webinar "Mitochondrial Functionality and Metabolism in Human T Cells", October 13, 2020

Staining recommendations

Considering phosphospecific flow cytometry experiments, one can follow the protocol published by Nolan et al, 2003.

Staining steps from the article:

- Use at least 106 cells for the analysis,

- Fix cells with 1.5% formaldehyde 10 min at RT (one can add 16% formaldehyde directly into the culture medium to obtain a final concentration of 1.5%).

- Permeabilize with ice-cold MeOH (resuspend the cells with vigorous vortexing in 500 μl ice‐cold MeOH).

- Incubate on ice for 10 min.

- Store at -20°C or continue with the protocol

- Wash cells 3 times with PBS/1% BSA then resuspended in staining media.

- Complete according standard protocol.

This protocol is described in details in the above mentioned publication: Krutzik, P. O. & Nolan, G. P. Intracellular phospho-protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry 55A, 61–70 (2003). PMID: 14505311 DOI: 10.1002/cyto.a.10072
Ready to use kits (BD CBA Cell Signalling Flex Set System) and solutions (BD Phosflow™ buffers) for staining of phosphorylated proteins are provided by Becton Dickinson. One can find useful information under the link below: