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

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
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