Cayman Cell-Based Assay Buffer B (10x); Qty- 25; Size-25 Ml

This assay utilizes proprietary human cells that provide constitutive expression of the Human type II Vascular Endothelial Growth Factor Receptor, referred to herein as VEGFR2, but also known as KDR and FLK-1.

VEGFR2, a member of the receptor tyrosine kinase (RTK) family, is highly expressed in vascular endothelial cells. VEGFR2 is a master regulator of the normal physiological processes of vasculogenesis, angiogenesis and lymphangiogenesis. Alternatively, dysregulated expression of VEGFR2 is strongly associated with cancer progression and tumor development.

There are several isoforms of the physiological signaling molecule vascular endothelial growth factor (VEGF); the 165aa isoform of VEGF-A is the reference activator provided with this kit. Stated simplistically, VEGF-A forms a complex with heparin (or heparan sulphate) and other co-receptors, which then bind to the extra-cellular domain of VEGFR2. Dimerization, activation of cytoplasmic tyrosine kinase domains and auto-phosphorylation ensue, followed by phosphorylation and activation of alternative associated intracellular signaling proteins.

Activated VEGFR2 may signal through several different pathways, the most prominent being Src, GRB2/Shc/SOS, PI3K, PLC-γ and JAK/STAT.1 These various signal transduction pathways culminate in the activation of pathway-related transcription factors and the induced expression of their respective target genes.

Phosphorylation and activation of the transcription factor NFAT is one outcome of VEGFR2 activation, and it is the signaling mechanism exploited by the reporter cells included in this kit. Accordingly, INDIGOs Reporter Cells contains an engineered luciferase reporter gene functionally linked to tandem NFAT genetic response element (GRE) sequences and a minimal promoter. Activated NFAT will bind to its corresponding GRE’s to initiate the formation of a complete transcription complex that drives Luc gene expression. Quantifying relative changes in luciferase activity in the treated reporter cells relative to the untreated cells provides a sensitive, dose-dependent surrogate measure of drug- or antibody-induced changes in VEGFR2 activity.

Considering the significant involvement of VEGFR2 in sustaining tumor development it continues to command much interest as a target for the development of novel, specific small molecule inhibitory drugs and antibodies. Accordingly, the primary application of this reporter assay is to screen test materials for any functional activity, either agonistic or inhibitory, that they may exert against VEGFR2 or the activator protein VEGF-A.

INDIGO’s Reporter Cells are transiently transfected and prepared as frozen stocks using a proprietary CryoMite™ process. This cryo-preservation method allows for the immediate dispensing of healthy, division-competent reporter cells into assay plates. There is no need for intermediate treatment steps such as spin-and-rinse of cells, viability determinations or cell titer adjustments prior to assay setup.

INDIGOs assay kits provide the convenience of an all-inclusive cell-based assay system. In addition to VEGFR2 Reporter Cells, provided are two optimized media for use in recovering the cryopreserved cells and for diluting test samples, the reference activator human VEGF-A (165 aa isoform, non-glycosylated), Luciferase Detection Reagents, and a cell culture-ready assay plate. [INDIGO Catalog Nos. IB15001 | IB15001-32 | IB15002]

WARNING This product is not for human or veterinary use.