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.
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