The components which make up Indigo Biosciences assay kits

View the slide viewer

Human VEGFR Reporter Assay Kit

Name: Human VEGFR Reporter Assay Kit
SKU: IB1500
Availability: InStock

SIZES SKU

1 x 96-well format assays IB15001

3 x 32 assays in 96-well format IB15001-32

1 x 384-well format assays IB15002

View All Product Documentation

Specifications
Data
Target Background
Documentation
Overview
Citation
Related Products

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). INDIGO’s VEGFR2 reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of VEGFR2. In addition to VEGFR2 Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human VEGFR2. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

Clear, Reproducible Results
All-Inclusive Assay Systems
Exceptional Cell Viability Post-Thaw
Consistent Results Lot to Lot

Product Specifications

Target Type		Growth Factor Receptor
Species		Human
Receptor Form		Native
Assay Mode		Antagonist
Kit Components		VEGFR Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) VEGF-A165, (ref. agonist; in PBS/0.1% BSA ) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate
Shelf Life		6 months
Orthologs Available		No
Shipping Requirements		Dry Ice
Storage temperature		-80C

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.

Target 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.