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

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Materials


Other materials required
HEK293 cell line expressing VSV-G epitope tagged β2-adrenergic receptor

HEK293 cell line expressing VSV-G epitope tagged δ-opioid receptor

HEK293 cell line expressing EGF receptor

HEK Culture medium: MEM (Sigma M2279) containing:
10% (v/v) FBS
Geneticin G418, 200 µg/ml (Sigma)
L-glutamine (Gibco)
Non-essential amino acids (Sigma)

Assay medium: MEM, phenol red free (Invitrogen 51200-038), or Krebs-Ringer buffer (see method)

Agonists: Isoproterenol, DADLE ([D-Ala2, D-Leu5]-Enkephalin), EGF (epidermal growth factor, murine submaxillary gland) (Sigma)

Hoechst 33342 (Molecular Probes)

Rat anti-human EGFR (Serotec, MCA1784)

Phosphate-buffered saline (PBS) solution

Poly-D-lysine, 5 mg (Sigma P6407)

96-well assay plate (Packard Viewplate)


Methods
Assay for β2-adrenergic and δ-opioid receptor internalization

1. Pre-coat 96-well plates with poly-D-lysine, recommended for HEK293 cell lines.

2. Seed cells at a density of ~ 16 000 cells/well.

3. Incubate ~ 48 h at 37 °C with 5% CO2.

4. Replace the culture medium with 100 µl/well of fresh MEM (phenol red free) containing 2.5–5 µg/ml CypHer5E anti-VSV-G antibody and 2.5 µM Hoechst.

5. Incubate at room temperature for 15 min.

6. Add 50 µl assay medium containing agonist and incubate at 37 °C for 30 min.

7. 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 o
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