iCyte™ Automated Imaging Cytometer

CompuCyte Corporation , Cambridge, MA, USA

Transfluor® Assay on the iCyte™ Imaging Cytometer

  1. Kazuo Ozawa, Ph.D., Etsuo Shinohara, and Sachiko Karaki, Ph.D.
  2. Olympus Corporation, Genome Medical Business Division, Tokyo, Japan
  3. Atto corporation


Transfluor® technology is a cell-based fluorescence assay system for screening G-protein coupled

receptors (GPCRs) developed by Norak Biosciences (Morrisville, NC, USA)1. The Transfluor assay

was developed based on the receptor desensitization mechanism. Upon ligand binding, GPCR is

activated by association with G protein and then desensitized by phosphorylation and arrestin binding followed by internalization and recycling. The desensitization mechanism is common to most GPCRs.

The Transfluor assay measures receptor activities by quantifying the redistribution of arrestin protein

from cytosol to GPCR on the cell

membrane and endosome. By labeling the

arrestin molecule with green fluorescence

protein (GFP), the Transfluor assay can

visually monitor the processes of

desensitization. Because the

desensitization is closely coupled to

activation of GPCR, changes in the

intracellular distribution of the arrestin

molecules reflect the active status of

GPCRs. By using a cell line transfected

with expression vectors of a particular

GPCR molecule and the arrestin-GFP, it is

possible to evaluate agonistic and

antagonistic activity of chemical

compounds towards specific GPCRs.

In the absence of agonist stimulation,

arrestin-GFP molecules are evenly

distributed throughout the cytosol

(Figure 1A).

Once GPCR is activated, intracellular

movement of the arrestin-GFP protein can

be observed under a fluorescence

microscope: toward clathrin-coated pit (Pittype signals, Figure 1B) within seconds; or

toward endocytic vesicles within minutes

(Vesicle-type signals, Figure 1C).

1A. Unstimulated

U-2 OS cells co-expressing arrestin-GFP plus wild-type

beta2-adrenergic receptor without receptor stimulation.

1B. Agonist-stimulated Pit-type signals

High-dose agonist-treated U2-OS cells co-expressing

arrestin-GFP and wild-type beta2-adrenergic receptor.

1C. Agonist-stimulated Vesicle-type signals

High-dose agonist-treated U2-OS cells co-expressing

arrestin-GFP and modified beta2-adrenergic receptor.

For information on this and other applications, email techsupport@compucyte.com

CompuCyte Corporation 12 Emily Street Cambridge MA 02139 USA Phone +1.800.840.1303 www.compucyte.com

Olympus Corporation 3-1 Nishi-Shinjuku 2-chome, Shinjuku-ku, Tokyo Japan 163-0914 www.olympus.com

Transfluor® Assay on the

iCyte™ Imaging Cytometer

Laser Scanning Cytometry

The iCyte™ Automated Imaging Cytometer, developed by CompuCyte Corporation (Cambridge, MA,

USA) is designed to meet the demand in the biotechnology and pharmaceutical markets for

automated high-content cellular analyzers which provide for higher throughput. iCyte has an

architecture suitable for quantitative evaluation of characteristics of compounds measuring biological

responses of cells and tissues. iCyte plays two roles: to acquire brightfield and fluorescent images of

cells and tissues automatically; then, to convert molecular and cellular biological states to numerical

data by analyzing these images within various parameters. These numerical data outputs are further

subjected to processing by statistical analysis software to calculate numerical indexes for quantitative

evaluation of chemical compounds.

Multi-well plates are often used to analyze cultured cells on iCyte. Because iCyte automatically

captures cell images in each well, it is possible to track gradual changes of cellular responses

corresponding to a dilution series of chemical treatment. Laser image scanning is carried out in a nonconfocal manner. Brightfield images are acquired by detecting scattered light with a special detector,

and fluorescent images are obtained by incorporating light emission detected by photomultiplier tubes

(PMT). Although slower than some camera-based systems, this optical architecture is superior in

terms of constituent quantitation.


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