( P96-well assay plate (P...)The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR

96-well assay plate (Packard Viewplate)

Method
Assay for β2-adrenergic, TRH, angiotensin II, and δ-opioid receptor internalization
1. Pre-coat 96-well plates with poly-D-lysine (recommended for HEK293 cells but not required for CHO [TRHR] cells).

2. Seed cells at a density of 10–15 000 cells/well.

3. Incubate overnight at 37 °C with 5% CO₂.

4. Replace the culture medium with either 100 µ l of fresh MEM (for β 2-adrenergic and δ -opioid receptor expressing cells) or 100 µ l Krebs-Ringer buffer (for TRH and angiotensin II receptor expressing cells) containing 5 µ g/ml of CypHer5 labelled anti-VSV-G antibody and 2.5 µ M Hoechst.

5. Incubate at room temperature for 15 min.

6. Add 100 µ l of assay medium or Krebs-Ringer buffer containing agonist and incubate at 37 °C for 30 to 40 min as appropriate (see Results for appropriate incubation time).

7. 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.

Conjugation of CypHer 5 Mono NHS Ester to anti-EGFR antibody
1. Commercially available antibodies may be supplied in buffer containing sodium azide. This can affect labelling efficiency and therefore should be removed by dialysis with PBS prior to labelling.

2. For details of the labelling method used, refer to the instructions supplied with CypHer5 Mono NHS Ester and/or the CypHer User Manual. For conjugation of CypHer5 Mono NHS Ester to anti-EGFR antibody, we recommend a 40 molar excess of CypHer5 Mono NHS Ester.

3. The final dye:protein (D:P) ratio for
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