Navigation Links
The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR


Introduction
CypHer5TM is a red-excited, pH-sensitive cyanine dye that is non-fluorescent at pH 7.4 and maximally fluorescent at pH 5.5. It is therefore ideally suited for monitoring internalization of cell surface receptors via acidic endosomal vesicles upon agonist stimulation (1, 2).

In the assay, the receptor can be epitope-tagged and an antibody recognizing the tag is then labelled with CypHer5. Alternatively, an endogenous antibody that recognizes an exofacial epitope can be labelled directly with CypHer5. Agonist-induced internalization of the receptor is then measured as an increase in fluorescence in the cell. This offers an advantage over other techniques that rely solely on measuring a translocation in fluorescent signal. Many other commonly used techniques such as b-arrestin recruitment, Ca2+ flux, and cAMP measurement are only suitable for use with G-protein coupled receptors (GPCRs). In contrast, CypHer5 is functional with both GPCRs and non-GPCRs that internalize in response to external stimuli.

GPCRs represent an important area for target validation. To demonstrate the generic applicability of CypHer5 pH-sensitive dye, examples of the GPCR subfamilies Gi (δ -opioid receptor), Gq (TRHR-1, angiotensin II) and Gs (β -2 adrenergic receptor) (3) were studied. These assays involve the use of CypHer5 labelled anti-VSV-G antibody to detect the VSV-G tagged receptors.

Epidermal growth factor receptor (EGFR) plays a central role in the complex signaling pathways involved in cell growth and differentiation (4) and is an example of a non-GPCR that is widely studied. The assay described here uses an exofacial antibody for EGFR that is directly labelled with CypHer5 Mono NHS Ester.


Products used
CypHer5 labelled anti-VSV-G antibody, 250 mg PA45407

CypHer5 NHS ester, 1 mg PA15401

IN Cell Analyzer 3000 25-8010-11

Granularity Analysis Module 63-0048-97
(for IN Cell Analyzer 3000)


Other materials required
HEK293 cell line expressing VSV-G epitope tagged β 2-adrenergic receptor

HEK293 cell line expressing VSV-G epitope tagged δ -opioid receptor

HEK293 cell line expressing VSV-G epitope tagged angiotensin II receptor

CHO cell line expressing VSV-G epitope tagged TRH 1 receptor

HEK293 cell line expressing EGF receptor

HEK Culture medium:
MEM (Sigma) containing 10% (v/v) FBS (Sigma),
200 µ g/ml Geneticin G418(Sigma),
L-glutamine (Gibco),
Non-essential amino acids (Sigma)

CHO culture medium:
HAM F12 Nutrient medium (Sigma) containing
10% (v/v) FBS (Sigma),
200 µ g/ml Geneticin G418 (Sigma),
L-glutamine, penicillin-streptomycin, (Gibco)

Assay medium: MEM, or Krebs-Ringer buffer (see method)

Agonists:
Isoproterenol, DADLE ([D-Ala2, D-Leu5]-Enkephalin),
TRH (thyrotropin releasing hormone), angiotensin II,
EGF (epidermal growth factor, murine submaxillary gland)
(Sigma)

Hoechst 33342 (Molecular Probes)

Rat anti-human EGFR (Serotec, MCA1784)

Phosphate buffered saline (PBS) solution

Poly-D-lysine, 5 mg (Sigma)

96-well assay plate (Packard Viewplate)


Method
Assay for β2-adrenergic, TRH, angiotensin II, and δ-opioid receptor internalization

1. Pre-coat 96-well plates with poly-D-lysine (recommended for HEK293 cells but not required for CHO [TRHR] cells).

2. Seed cells at a density of 10–15 000 cells/well.

3. Incubate overnight at 37 °C with 5% CO2.

4. Replace the culture medium with either 100 µ l of fresh MEM (for β 2-adrenergic and δ -opioid receptor expressing cells) or 100 µ l Krebs-Ringer buffer (for TRH and angiotensin II receptor expressing cells) containing 5 µ g/ml of CypHer5 labelled anti-VSV-G antibody and 2.5 µ M Hoechst.

5. Incubate at room temperature for 15 min.

6. Add 100 µ l of assay medium or Krebs-Ringer buffer containing agonist and incubate at 37 °C for 30 to 40 min as appropriate (see Results for appropriate incubation time).

7. Image on the IN Cell Analyzer 3000 with the following filter sets: 633 nm excitation and 695BP55 emission for CypHer5, 365 nm excitation and 450BP65 emission for Hoechst.


Conjugation of CypHer 5 Mono NHS Ester to anti-EGFR antibody
1. Commercially available antibodies may be supplied in buffer containing sodium azide. This can affect labelling efficiency and therefore should be removed by dialysis with PBS prior to labelling.

2. For details of the labelling method used, refer to the instructions supplied with CypHer5 Mono NHS Ester and/or the CypHer User Manual. For conjugation of CypHer5 Mono NHS Ester to anti-EGFR antibody, we recommend a 40 molar excess of CypHer5 Mono NHS Ester.

3. The final dye:protein (D:P) ratio for CypHer5 labelled anti-EGFR was calculated to be 1.4:1 (the CypHer5 anti VSV-G antibody uses a D:P ratio of between 1.5:1 and 3.0:1 for optimum performance).

4. Labelled antibody should immediately be diluted to 0.5 mg/ml in PBS containing 0.1% BSA, centrifuged to remove any precipitate, dispensed into suitable aliquots, and stored at -15 to -30 °C. Avoid repeated freeze-thaw cycles and protect from light.


Assay for EGF receptor internalization
1. Pre-coat 96-well plates with poly-D-lysine (recommended for HEK293 cells).

2. Seed cells at a density of 10 000 cells/well.

3. Incubate for 48 h at 37 °C with 5% CO2.

4. Replace culture medium with serum-free medium and continue incubation for at least 4 h prior to assay.

5. Replace the medium with 50 µ l of Krebs-Ringer buffer containing 5 µ g/ml of CypHer5 labelled anti-EGFR antibody and 2.5 µ M Hoechst.

6. Incubate at room temperature for 15 min.

7. Add 50 µ l of Krebs-Ringer buffer containing EGF and incubate at 37 °C for 30 min.

8. Image on the IN Cell Analyzer 3000 with the following filter sets: 633 nm excitation and 695BP55 emission for CypHer5, 365 nm excitation and 450BP65 emission for Hoechst.


Results
Images show the accumulation of CypHer5 as a perinuclear clustering of red fluorescence for the δ -opioid and EGF receptors upon ligand activation (Fig 1). This can be compared to the minimal fluorescence observed prior to agonist addition. Fluorescence from Cypher5 is localized to acidic endosomes as a result of agonist-induced internalization of the receptor-ligand complex and is observed for both a GPCR (δ -opioid receptor) and non-GPCR (EGFR).

The r esponse was quantitated using the Granularity Analysis Module for the IN Cell Analyzer 3000, which identifies, counts, and analyzes the grain-like fluorescent structures within cells. A typical analysis of the response for EGFR is shown in Figure 2.

Agonist-induced internalization of CypHer5 labelled antibodies is concentration dependent. A sigmoidal dose response curve was obtained for each agonist by non-linear regression analysis. The dose response curves produced for EGFR and for each of the VSV-G-tagged β 2-adrenergic, δ -opioid, angiotensin II, and TRH receptors are shown in Figure 3. The VSV-G epitope tag provided improved results using CypHer5 when compared to those obtained with a c-myc epitope tag (data not shown, for further information contact incellanalyzer@amersham.com). The calculated EC50 values were comparable to published data, where available (5).


Conclusion
In principle, CypHer5 pH-sensitive dye can be applied to the functional analysis and screening of any receptors that internalize into acidic endosomal vesicles upon ligand binding and activation. Dose response data were obtained for a range of different GPCR subtypes, producing acceptable EC50 values. In addition it has been demonstrated that CypHer5 can be used to study the internalization of non-GPCRs. As an example, dose response data were generated for the tyrosine kinase receptor for EGF. Furthermore, for this assay, the use of a CypHer5 labelled antibody that binds an exofacial epitope of EGFR (rather than a tag) was shown. The data show that assays using CypHer5 pH-sensitive dye can be performed on the IN Cell Analyzer 3000 although pharmacologically relevant data can also be produced using the IN Cell Analyzer 1000 or a range of confocal microscopes.


References
1. Adie, E.J., et al., BioTechniques, 33, 1152 (2002).
2. Milligan, G., Drug Discovery Today, 8, 579–585 (2003).
3. Application note: Screening for β2-adrenergic receptor agonists using the pH-sensitive dye, CypHer5, and the IN Cell Analyzer 3000, Amersham Biosciences, 11-0002-81, Edition AA (2003).
4. Kim, J. et al. Biochemistry, 42, 2887–2894 (2003).
5. Oakley, R.H. et al. Journal of Biological Chemistry, 274, 32248–32257 (1999).

Note: The CypHer5 dye used in this application note was replaced in December 2003 by CypHer5E. This new pH-sensitive cyanine dye has the same mechanism of action as the previous compound but with the following enhanced features:

1. Higher extinction coefficient: can be used with cell lines that have lower receptor expression levels, enabling assays to be imaged on lower sensitivity instruments.

2. Increased sulfonation: Improved solubility, greater labeling efficiencies, and improved stability in solution.

More information comparing the performance of the two dyes can be found in the application note: The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies (11-0008-22AA).


For further information, please contact your local sales office:


back to top
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000
2. The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies
3. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
4. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
5. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
6. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
7. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
8. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
9. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors
10. Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
11. The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/23/2016)... , ... June 23, 2016 , ... ... quality, regulatory and technical consulting, provides a free webinar on Performing ... July 13, 2016 at 12pm CT at no charge. , Incomplete investigations are ...
(Date:6/23/2016)... 22, 2016  Amgen (NASDAQ: AMGN ) ... QB3@953 life sciences incubator to accelerate the development ... laboratory space at QB3@953 was created to help high-potential ... for many early stage organizations - access to laboratory ... Amgen launched two "Amgen Golden Ticket" awards, providing each ...
(Date:6/22/2016)... Cell Applications, Inc. and StemoniX announced ... up to one billion human induced pluripotent stem ... These high-quality, consistent stem cells enable researchers to ... more time doing meaningful, relevant research. This achievement ... process that produces affordable, reliable HiPSC for life ...
(Date:6/22/2016)... YORK , June 22, 2016  According ... growing next generation sequencing (NGS) market include significant ... of smaller sequencers.  More accessible and affordable sequencers, ... to growing demand for consumables including sample prep ... The Market for Sample Preparation for Next Generation ...
Breaking Biology Technology:
(Date:6/2/2016)... LONDON , June 2, 2016 ... Systems, Manned Platforms, Unmanned Systems, Physical Infrastructure, Support & ... intelligence provider visiongain offers comprehensive analysis of ... that this market will generate revenues of $17.98 billion ... Systems acquired DVTEL Inc, a leader in software and ...
(Date:5/16/2016)... NEW YORK , May 16, 2016   ... authentication solutions, today announced the opening of an IoT ... to strengthen and expand the development of embedded ... provides an unprecedented level of convenience and security with ... to authenticate one,s identity aside from DNA. EyeLock,s platform ...
(Date:4/28/2016)... India , April 28, 2016 ... Infosys (NYSE: INFY ), and Samsung SDS, a ... that will provide end customers with a more secure, ... services.      (Logo: http://photos.prnewswire.com/prnh/20130122/589162 ) , ... services, but it also plays a fundamental part in enabling ...
Breaking Biology News(10 mins):