At its core, cell signaling is mediated through the modulation of specific proteins. Phosphorylation is the addition of a phosphate group to the polar group on various amino acids- principally, serine, threonine, and tyrosine through the activity of highly specific kinase enzymes. The opposite reaction, dephosphorylation, is the removal of a phosphate group via a phosphatase enzyme.
Within the human proteosome, there are over
200,000 known phosphorylation sites
and up to 30% of proteins may be modified through kinase and phosphatase activity.
These include a diverse array of functional proteins from membrane-bound receptors, ion channels, and transporters, to cytosolic enzymes, cell signaling, protein degradation associated proteins, and nuclear transcription factors and histones.
When it comes to interrogating cell signaling mechanisms, phosphoflow provides unparalleled granularity, enabling researchers to decipher the responding cell populations and monitor these mechanisms in detail.
There are a series of proteins that are transiently phosphorylated within a signaling pathway, and so understanding the timing of these events is critical if they are to be detected with phosphoflow before either dephosphorylation or protein degradation is triggered.
Many of the target proteins have multiple potential phosphorylation sites, that may be regulated very differently. Make sure that your antibody conjugate is targeting the correct epitope for the signal-specific phosphorylation. Always ensure that any conjugate used for any flow cytometry application have been validated for this use.
Phosphoflow requires the fixation and permeabilization of the cell. Permeabilization is a balance of gaining access to the intracellular compartment, versus the loss of protein targets. Fixation conditions can impact the landscape of the cell for phosphoflow detection. All of these parameters should be evaluated for their effect on activation specific phospho-signal.
In many cases, phosphoflow is performed in conjunction with CD marker staining to identify specific cell populations. It is important to carefully access the binding of conjugates to their target epitopes under different processing conditions- CD cell lineage staining may be performed prior to fixation, or after permeabilization.
Unstimulated negative and stimulated positive controls, as well as compensation controls (or BD CompBead Particles) are all recommended. As will all flow analysis, a viability dye should be used: fixable viability dyes such as Ghost dyes are a great option for phosphoflow.
Baselines for phosphoflow analysis can be tricky, so it is important to evaluate and be consistent with pre-stimulation conditions -although cells shouldn’t be starved, changing media the day of stimulation can alter signaling, and cells should be rested for ~90 minutes at 37˚C before stimulation.
Profile cell signaling responses to lead compounds
Direct analysis of up- and down-regulation of pathways in animal models
Establish cell signaling profiles for therapeutic targeting
Multiplexed signaling pathway analysis
In vitro drug safety and efficacy screening
Fluorescent Cellular Barcoding
In vivo drug efficacy and safety screening
Cellular assays to monitor clinical endpoints
Support PK/PD
Mechanism of Action
Identify disease-specific cellular responses
Application of precision medicine approaches to disease management
Implement prognostic markers
STAY CONNECTED