8. Analyze the images using the Granularity Analysis Module.
Conjugation of CypHer5E NHS ester to anti-EGFR antibody
1. Commercially available antibodies may be supplied in buffer containing sodium azide. This can affect labeling efficiency and therefore should be removed by dialysis with PBS prior to labeling.
2. For details of the labeling method used, refer to the instructions supplied with CypHer5E NHS ester (PAI5401) or the CypHer User Manual. For conjugation of CypHer5E NHS ester to anti-EGFR antibody we recommend a 20 M excess of CypHer5E NHS ester.
3. The final dye:protein (D:P) ratio for CypHer5E-labeled anti-EGFR was calculated to be 5.8. For information, the CypHer5E anti-VSV-G antibody (PA45407) uses a D:P ratio of between 7.0 and 12.0 for optimum performance. (Note: there is an increase in D:P ratio with CypHer5E compared to CypHer5 due to the enhanced characteristics of the new dye such as solubility and stability).
4. Labeled 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 ~ 16 000 cells/well.
3. Incubate ~ 48 h at 37 °C with 5% CO2.
4. Replace culture medium with serum-free medium and continue incubation for at least 4 h (or overnight) prior to assay.
5. Replace the medium with 50 µl Krebs-Ringer buffer containing 2.5–5 µg/ml CypHer5E labeled anti-EGFR antibody and 2.5 µM Hoechst.
6. Incubate at room te