The phosphorylation and dephosphorylation of key regulatory signaling proteins govern basic cellular responses to their microenvironment. Since dysfunction of these signaling pathways is an underlying cause of many diseases, these regulatory proteins are now being targeted for therapeutic intervention. The critical analysis of the state of these regulatory signaling proteins by flow cytometry is referred to as phosphoflow, a technique that provides unprecedented characterization of signaling pathway activation and deactivation and resulting the downstream functional responses, within heterologous cell samples.
Using phosphoflow it is possible to identify the regulation of signaling pathways at the single-cell level, to provide high resolution comparative analysis of phenotypic and functional responses for targeted drug development.
Figure 1. PhosphoFlow Staining Technique for Flow Cytometry.
(1) Heterogeneous sample of cells are treated with two different stimuli, A and B, or a combination of the two A+B to induce phosphorylation of target proteins. (2) Cells are then fixed, permeabilized and stained. (3) Cells are analyzed on a flow cytometery
Strategic experimental design to ensure optimal cell activation, and post-fixation, permeabilization and staining.
Library of qualified phosphoflow protocols to support your safety, efficacy, and mechanism of action studies.
High-resolution of complex signaling events to uncover the activation of distinct pathways, even within rare cell types.
Figure 2. P-STAT-5 Activation Profiles. Which Lymphocyte Respond to IL-2?
Activation Profiles for a. CD4+, b. CD8+ T Cells, and c. CD19+ B Cells. The data is displayed as a histogram overlays of the MFI signaling profiles of the phosphorylation marker STAT-5: unstimulated cells (solid grey), and after stimulation with 100ng/ml IL-2 (black outlined white peaks). The data was collection from human whole blood samples. This analysis demonstrates the T-cell responses to IL-2 includes phosphorylation of STAT-5. This response it not observed in the B-cells.
The Scientific Team at FlowMetric has extensive experience in developing qualified fit-for-purpose flow cytometry panels to examine various aspects of phosphorylation-mediated cellular responses. We provide advanced platforms that enable us to obtain comprehensive data sets, ensuring full support of your pre-clinical and clinical trial needs.