( CypHer5 labelled anti-EGFR was calculat...)The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR

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).

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