Multiparameter flow cytometry offers key advantages for detection and analysis of intracellular signaling events within cellular subsets in complex cell mixtures. This detection of phosphorylated proteins by flow cytometry is often referred to as phosphoflow assays (Phosflow™ Assay). Phosphosflow assays are used to examine cellular responses to treatment with stimulatory/inhibitory molecules or drug entities.
With the ability to collect data at the level of individual cells, flow cytometry offers several advantages for detecting various phosphorylated proteins in cell signaling studies. Unlike lysate-based approaches, phosphoflow assays facilitate the detection and analysis of heterogeneous signaling responses in complex biological matrices. With the addition of antibodies specific for cell surface markers to cellular subsets within complex cell mixtures such as whole blood, signaling responses mediated by protein phosphorylation can be detected. Moreover, rare cell populations can be uncovered without pre-enrichment of cells. As a result, rich data is obtained from limited cell samples.
While other techniques can measure either cytokines, transcription factors or phosphorylated proteins separately, intracellular flow cytometry enables measurement of multiple intracellular markers simultaneously at the single cell level. This provides data on signaling responses, differentiation states and other cellular events. The combined use of antibodies specific for cell surface and intracellular markers enables high resolution comparative analysis of the phenotypic and functional differences with multiple cell types across samples.