Features

• Validated with over 100 GPCRs, the Transfluor assay provides the assurance to work across all GPCR classes (Class I, II, III), regardless of interacting G-protein (Gs, Gi/o and Gq/11).
• Single read-out is compatible with all GPCR subtypes, including Gi, Gs, Go, Gq, eliminating the need for multiple GPCR assays
• No GPCR labeling or tagging avoids custom molecular biology that may alter your target's response.
• Universal format requires no prior knowledge of interacting G-protein, enabling orphan GPCR screening.
• GFP-based technology does not require additional substrates to monitor beta-arrestin translocation.
• MetaXpress® Application Module automates analysis of beta-arrestin movement to the cell membrane (pits) and endocytic vesicles.
• Ligand Independent Translocation (LITe™) Assay is an agonist-independent assay used to verify the translocation of beta-arrestin-GFP in orphan GPCRs.

Technology Overview

With the Transfluor technology, arrestin-GFP is dispersed throughout the cytosol in unstimulated cells. Upon ligand binding with the GPCR, the arrestin-GFP quickly translocates from the cytosol to the cell membrane and then into clathrin-coated pits at the membrane. The receptor then internalizes into endocytic vesicles prior to reprocessing and returns to the cell membrane. Pits and vesicles can be imaged and quantified by commercially-available image analysis instrumentation, such as ImageXpress® Micro or ImageXpress® Ultra System, forming the basis for a universal, high-throughput, high-content assay for all GPCRs (known and orphan).

View a Transfluor technology video (4 MB MOV; requires QuickTime)

View an animation of the desensitization process and how Transfluor works (10 MB SWF; requires Flash)

Download a free copy of the QuickTime player from the Apple web site.
Download a free copy of the Flash player from the Macromedia web site.

For best results, you should have a high-speed connection to the Internet. If you do not have a high-speed connection, the animation or video may take several minutes to start.

Orphan GPCR Assay

In contrast to current methods of screening GPCRs, the Transfluor technology is based on the mechanism for termination of GPCR signaling, known as receptor desensitization. This mechanism is shared by virtually all GPCRs and is activated by ligand binding. Transfluor technology requires no prior knowledge of the interacting G-protein. This important feature of the Transfluor technology makes it ideal for screening orphan GPCRs (oGPCR).

A proprietary technique to assist in validating orphan GPCR screens, called LITe™ (Ligand Independent Translocation), is an agonist-independent assay used to verify the translocation of ß arrestin-GFP in orphan GPCRs.

Transfluor References

Garippa RJ, Hoffman AF, Gradl G, Kirsch A.
High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.
Methods Enzymol. 2006;414:99-120.

Hudson CC, Oakley RH, Sjaastad MD, Loomis CR.
High-content screening of known G protein-coupled receptors by arrestin translocation.
Methods Enzymol. 2006;414:63-78.

Oakley RH, Hudson CC, Sjaastad MD, Loomis CR.
The ligand-independent translocation assay: an enabling technology for screening orphan G protein-coupled receptors by arrestin recruitment.
Methods Enzymol. 2006;414:50-63.

Haasen D, Wolff M, Valler MJ, Heilker R.
Comparison of G-protein coupled receptor desensitization-related beta-arrestin redistribution using confocal and non-confocal imaging.
Comb Chem High Throughput Screen. 2006 Jan;9(1):37-47.

Ghosh RN, DeBiasio R, Hudson CC, Ramer ER, Cowan CL, Oakley RH.
Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.
J Biomol Screen. 2005 Aug;10(5):476-84.

Ozawa K, Hudson CC, Wille KR, Karaki S, Oakley RH.                               Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.
Cytometry A. 2005 May;65(1):69-76.

Oakley RH, Hudson CC, Cruickshank RD, Meyers DM, Payne RE Jr, Rhem SM, Loomis CR.
The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.
Assay Drug Dev Technol. 2002 Nov;1(1 Pt 1):21-30.

Partial GPCR Listing

The Transfluor technology monitors receptor activity by detecting movement of beta-arrestin-GFP in the cell. A partial listing of GPCRs that have been shown to translocate beta-arrestin-GFP is shown below.

Other Receptor Listing

• 12 Drosophila GPCRs
• Fz4 frizzled receptor
• TßRIII transforming growth factor-ß

Molecular Devices Transfluor Cell-Based GPCR Assay is compatible with the ImageXpress Micro Widefield and ImageXpress Ultra Confocal high content screening systems, which rapidly acquire high resolution images for research and discovery applications. 

The Transfluor® Application Module for MetaXpress® Software is available to facilitate analysis of receptor internalization specifically for the Transfluor assay and is scalable for high-speed image analysis using MetaXpress® PowerCore High Content Distributed Analysis Software.

Ordering Information

Purchase of these Transfluor products requires a prior execution of the Transfluor License agreement and the BioImage license. If ordering the GFP cell line or plasmid, a Prolume license is also required for purchase.

Transfluor Evaluation Kit

The Transfluor Evaluation Kit enables use of the technology for a limited, one-time, six month evaluation period.  The kit provides all the necessary tools for evaluating the technology with the users' own instrumentation and GPCRs. This allows users to become comfortable with the technology, before proceeding to a tiered licensing option that meets users' specific needs.

Transfluor Evaluation Kit Components

• U-2 OS β-arrestin2-GFP cells (Parental cell line, to assist with determination of the Transfluor response to end-users' choice of GPCR)
• Transfluor Demo Plate (Imaging  grade  plate with fixed cells to assist with instrument validation and image acquisition and analysis)
• Limited Label License
• Transfluor Evaluation Kit  Product Insert
• U-2 OS β-arrestin2-GFP cells Product Insert
• Transfluor Quick Start Protocol

Transfluor User Guide (complete with protocols and troubleshooting guidelines)

Please contact your local sales representative for detailed information regarding this kit.

Tiered Licensing Options

Use of the Transfluor technology requires the purchase of a Transfluor Technology Use License from Molecular Devices (MOLDEV) which is renewable on an annual basis. A scalable license structure is available from research up to high-throughput screening including the following examples:

• Transfluor Assay Research Site License
• Transfluor Assay Research Global License
• Transfluor Screening Site License
• Transfluor Assay Research and Screening Site License

Two additional third-party licenses are required to run the Transfluor assay: a GFP license (MOLDEV recommends the Prolume Renilla reniformis (Rr) GFP license) and BioImage's fluorescence redistribution license. Terms are as follows:

• Prolume license is renewable on an annual basis for a nominal fee.
• BioImage license is a one-time fee.
• Prolume and BioImage licenses both provide worldwide coverage.

To streamline your process, you can obtain these licenses directly from MOLDEV. Alternatively, you can also obtain them directly from Prolume and BioImage.

Please contact your local sales representative for detailed information regarding these licenses. In conjunction with the Transfluor license, MOLDEV also offers a stably transfected GFP beta-arrestin starter cell line and relevant plasmids.