Cy3-Transferrin

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
1.Serum Starvation: To increase the receptor capacity for labeled transferrin, starve cells in a serum-free medium for 30-60 minutes at 37°C.
2.Ligand Preparation: Prepare a working solution of Cy3-Transferrin. A common concentration is 10-50
g/mL in binding buffer.
3.Binding/Uptake (Internalization):
3.1 Option A (Active Endocytosis): Replace the medium with the pre-warmed Cy3-Transferrin solution and incubate at 37°C for 5–30 minutes to allow internalization.
3.2 Option B (Surface Binding + Pulse-Chase): Incubate with Cy3-Transferrin for 15–30 minutes on ice (4°C) to allow binding without endocytosis. Wash 3x with cold buffer, then move to 37°C to initiate internalization (chase).
4. Washing: Move the cells to an ice bath immediately and wash 3–4 times with cold buffer to remove free, unbound ligand.
5. Fixation: Fix the cells with 4% PFA in PBS for 10-15 minutes at room temperature, then wash with PBS.
6. Imaging/Analysis: Observe cells using confocal fluorescence microscopy. Internalized Cy3-Transferrin will appear as red fluorescent puncta (early/recycling endosomes).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.