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The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies


Introduction
Measurement of receptor internalization using the pH-sensitive cyanine dye, CypHer™5, has previously been reported (1,2,3). An enhanced version of this dye that shows greater relative fluorescence intensity when maximally fluorescent, greater solubility, and stability has now been developed. The enhanced dye, CypHer5E, is a red-excited, pH-sensitive fluorogenic dye that is non-fluorescent at basic pH and maximally fluorescent at acidic pH. This enables the monitoring of cell surface receptors internalizing into acidic endosomal vesicles upon agonist stimulation (4,5).

Using N-terminal VSV-G tagged receptors and CypHer5E-labeled anti-VSV-G antibodies, receptor internalization can be monitored. Alternatively a CypHer5E-labeled antibody recognizing an endogenous epitope may be used. Agonist treatment produces an increase in fluorescence when the CypHer5E is internalized in association with the receptor. This increase in fluorescence can be measured on a range of fluorescence microscopes, or platforms such as the automated lamp-based IN Cell Analyzer 1000, with integrated image analysis software.

GPCRs are the largest family of proteins involved in transmembrane signal transduction and represent an important area for the validation of therapeutic small molecule drug targets. Agonist-induced internalization of CypHer5E-labeled antibodies was observed for all G-protein sub classes of GPCR (Gi, Gs, and Gq) and for a non-GPCR receptor, tyrosine kinase.

Here we present data using the β-2 adrenergic, δ-opioid, and EGF receptors in live-cell model assays to demonstrate the use of CypHer5E enhanced dye and the IN Cell Analyzer 1000 for receptor internalization studies. CypHer5E generates pharmacologically relevant data and enables internalization to be measured in cell lines with lower receptor expression levels than was previously possible with CypHer5 dye.


Materials


Other materials required
HEK293 cell line expressing VSV-G epitope tagged β2-adrenergic receptor

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

HEK293 cell line expressing EGF receptor

HEK Culture medium: MEM (Sigma M2279) containing:
10% (v/v) FBS
Geneticin G418, 200 µg/ml (Sigma)
L-glutamine (Gibco)
Non-essential amino acids (Sigma)

Assay medium: MEM, phenol red free (Invitrogen 51200-038), or Krebs-Ringer buffer (see method)

Agonists: Isoproterenol, DADLE ([D-Ala2, D-Leu5]-Enkephalin), 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 P6407)

96-well assay plate (Packard Viewplate)


Methods
Assay for β2-adrenergic and δ-opioid receptor internalization

1. Pre-coat 96-well plates with poly-D-lysine, recommended for HEK293 cell lines.

2. Seed cells at a density of ~ 16 000 cells/well.

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

4. Replace the culture medium with 100 µl/well of fresh MEM (phenol red free) containing 2.5–5 µg/ml CypHer5E anti-VSV-G antibody and 2.5 µM Hoechst.

5. Incubate at room temperature for 15 min.

6. Add 50 µl assay medium containing agonist and incubate at 37 °C for 30 min.

7. Image on the IN Cell Analyzer 1000 (20× objective), exposure time 300 ms/field, 620 nm excitation and 700 nm emission for CypHer5E; exposure time o f 500 ms/field, 360 nm excitation and 535 nm emission for Hoechst.

8. Analyze the images using the Granularity Analysis Module.


Conjugation of CypHer5E NHS ester to anti-EGFR antibody
1. Commercially available antibodies may be supplied in buffer containing sodium azide. This can affect labeling efficiency and therefore should be removed by dialysis with PBS prior to labeling.

2. For details of the labeling method used, refer to the instructions supplied with CypHer5E NHS ester (PAI5401) or the CypHer User Manual. For conjugation of CypHer5E NHS ester to anti-EGFR antibody we recommend a 20 M excess of CypHer5E NHS ester.

3. The final dye:protein (D:P) ratio for CypHer5E-labeled anti-EGFR was calculated to be 5.8. For information, the CypHer5E anti-VSV-G antibody (PA45407) uses a D:P ratio of between 7.0 and 12.0 for optimum performance. (Note: there is an increase in D:P ratio with CypHer5E compared to CypHer5 due to the enhanced characteristics of the new dye such as solubility and stability).

4. Labeled 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 ~ 16 000 cells/well.

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

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

5. Replace the medium with 50 µl Krebs-Ringer buffer containing 2.5–5 µg/ml CypHer5E labeled anti-EGFR antibody and 2.5 µM Hoechst.

6. Incubate at room te mperature for 15 min.

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

8. Image on the IN Cell Analyzer 1000 (20× objective), exposure time 300 ms/field, 620 nm excitation and 700 nm emission for CypHer5E; exposure time of 500 ms/field 360 nm excitation and 535 nm emission for Hoechst.

9. Analyze the images using the Granularity Analysis Module.


Results
IN Cell Analyzer 1000 images of the live-cell β-2 adrenergic assay show agonist induced internalization of CypHer5E into acidic endosomes (Fig 1). Although trafficking destinations are dependent on the receptor, similar patterning was observed with the δ-opioid and EGF receptors (data not shown). In the absence of ligand activation by an agonist, there is very little cell-associated fluorescence (Fig 1a). Gray-scale images show a perinuclear clustering of fluorescent granules in the red channel, which results in an increase in the red signal intensity (Fig 1b). A fused image showing the nucleus (blue) and CypHer5E (red) signal demonstrates the juxtanuclear localization of CypHer5E, indicative of a recycling endosome compartment (Fig 1c).

Agonist-induced internalization of CypHer5E-labeled antibodies is concentration dependent. The internalization was quantitated using the IN Cell Analyzer 1000 Granularity Analysis Module. The analysis routine identifies, counts, and analyzes the grain-like CypHer5E fluorescent structures within cells. A sigmoidal dose response curve was obtained for each agonist by non-linear regression analysis. Dose response curves for the β-2 adrenergic, δ-opioid, and EGF receptors are shown in Figure 2. Calculated EC50 values are comparable to values previously obtained with CypHer5 dye and with other literature values (6).

Receptors of interest will be expressed at the cell membrane at va rying levels dependent on factors such as receptor, cell type, and trafficking. The signal intensity increase observed with CypHer5E, compared to CypHer5, enables the monitoring of poorly expressed receptors. An example of this is shown in Figure 3. In addition to the δ-opioid cell line shown in Figure 2b (δ-opH), a lower expressing δ-opioid cell line was also generated (δ-opL). Expression levels of the two cell lines were determined by flow cytometry analysis. Signal to noise (S:N)* values were compared for four series of values (n = 4), each at the plateau for both CypHer5E and CypHer5. An average S:N value of 11.86 was obtained with CypHer5E, compared to 5.09 with CypHer5. The increased intensity and S:N values observed for CypHer5E with δ-opL indicate that the enhanced dye can compensate for the greater variation seen with lower expressors. Therefore users can still develop a robust assay. This is of particular benefit in assay development for screening applications. CypHer5E will provide the capability of monitoring the activation of lower expressing receptors than CypHer5. The data from Figure 3 also demonstrates very similar EC50 values for the same receptors at different expression levels.


Conclusion
CypHer5E is an enhanced pH-sensitive dye that can be applied to the functional analysis and screening of receptors that internalize into acidic endosomal vesicles upon ligand binding and activation. Using the IN Cell Analyzer 1000, a high-throughput lamp based sub-cellular imaging system, dose response data were obtained for a range of different GPCRs and a non-GPCR subtype. CypHer5E can be used to label antibodies that recognize an exofacial epitope on the target receptor, either endogenous to the protein or an inserted tag, the results are pharmacologically comparable to those obtained with CypHer5. The signal obtained with CypHer5E is significantly increased over that of CypHer5, thereby providing an increased enablement of the dye for monitoring receptors that have reduced epitope availability at the extracellular environment, either due to lower expression levels or trafficking irregularities. These results in conjunction with other data from the IN Cell Analyzer 3000 (laser based platform), demonstrate generic applicability of CypHer5E, not only with respect to cell membrane receptors, but also across a range of microscopy based systems.


References
1. The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR, Amersham Biosciences, 11-0002-80, Edition AA (2004).

2. Screening for beta 2-adrenergic receptor agonists using the pH-sensitive dye, CypHer5, and the IN Cell Analyzer 3000, Amersham Biosciences, 11-0002-81, Edition AA (2004).

3. Pharmacological characterization of beta 2-adrenergic receptor with pH-sensitive dye, CypHer5, and IN Cell Analyzer 3000, Amersham Biosciences, 11-0002-82, Edition AA (2004).

4. Adie, E.J. et al. BioTechniques 33, 1152 (2002).

5. Milligan, G. Drug Discovery Today 8, 579-585 (2003).

6. Oakley, R.H. et al. Journal of Biological Chemistry 274, 32248-32257 (1999).


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