Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)
|Product Family||Product Number||Product Description||Technical Manual|
|IB15001-32||Human VEGFR2 Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB15001||Human VEGFR2 Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB15002||Human VEGFR2 Reporter Assay System, 1 x 384-well format assays|
Human Vascular Endothelial Growth Factor Receptor 2 Assay Kit
This Human type II Vascular Endothelial Growth Factor Receptor (VEGFR2) assay kit is an all-inclusive firefly luciferase reporter assay system that includes in addition to VEGFR2 Reporter Cells, 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.
VEGFR2 Reporter Cells are transiently transfected and prepared as frozen stocks using INDIGO's 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 cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup.
INDIGO’s VEGFR2 assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
VEGFR2 reporter assay kits are offered in different assay formats to accommodate researchers' needs: 3x 32, 1x 96, and 1x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
Human Vascular Endothelial Growth Factor Receptor 2 Assay Services
The primary application of INDIGO's cell-based VEGFR2 assays are to quantitatively assess the bioactivity of a test compound as an agonist or antagonist of the receptor. Service assays include a positive control reference compound and 'vehicle' control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online "Request a Quote" form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Human Vascular Endothelial Growth Factor Receptor 2 Assay Background
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 vascular endothelial growth factor receptor type II (VEGFR2). Dimerization, activation of cytoplasmic tyrosine kinase domains and auto-phosphorylation ensue, followed by phosphorylation and activation of alternative associated intracellular signaling proteins.
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.
Activated VEGFR2 may signal through several different pathways, the most prominent being Src, GRB2/Shc/SOS, PI3K, PLC-γ and JAK/STAT. 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, INDIGO's 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.
This assay utilizes proprietary human cells that provide constitutive expression of the Human type II Vascular Endothelial Growth Factor Receptor (VEGFR2), also known as KDR and FLK-1.
Synonyms: Vascular Endothelial Growth Factor Receptor, VEGFR, Human type II Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Receptor 2, VEGFR2
Human Vascular Endothelial Growth Factor Receptor 2 Assay Data
VEGFR2 Activation assay. The VEGFR2 activation assay was performed according to the protocol provided in this Technical Manual. 200 µl/well of VEGFR2 Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. The reference peptide, non-glycosylated VEGF-A165, was further diluted using CSM+H to generate the desired assay concentrations of treatment media, as depicted in APPENDIX 1. The pre-culture media were discarded from the assay wells and 200 µl/well of treatment media were dispensed (n = 4 / conc.), including ‘untreated’ control wells. ‘Mock’ reporter cells (not included in this kit) contain the NFAT-Luc reporter gene but lack expression of VEGFR2. Following a 22 hr incubation period treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of standard deviation (SD), percent coefficient of variation (%CV) and Z’ were determined. Non-linear regression analyses of Fold-Activation vs. VEGF-A Log10[ng/mL] and EC50 determination were performed using GraphPad Prism software; error bars depict +/- %CV.
VEGFR2 Inhibition assays. 200 µl/well of VEGFR2 Reporter Cell suspension was dispensed into 96-well assay plates, which were then pre-incubated for 4 hours. For inhibition-mode assays, CSM+H was supplemented with 1.5 ng VEGF-A/ml. The small molecule inhibitors BMS 605541, Ki8751, ZM323881 and XL184 (Tocris) were initially prepared as concentrated stocks in DMSO, then further diluted using the VEGF-A165 supplemented CSM+H. Pre-culture media were discarded and 200 µl/well of the prepared treatment media were dispensed (n = 4 / conc.), including ‘vehicle only’ control wells. After 22 hr incubation the treatment media were discarded, Luciferase Detection Reagent was added, and RLU/well were quantified. Plots are RLU vs. VEGF-A Log10[nM]; error bars depict +/- SD.