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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.

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