Cy3-Transferrin is a fluorescent labeling reagent that combines Cy3 fluorescent dye and Transferrin. The Cy3 fluorophore is commonly used in applications such as immunolabeling, nucleic acid labeling, fluorescence microscopy, and flow cytometry. Cy3 has an emission maximum around 562-570 nm. Transferrin, a blood-plasma glycoprotein, is a target ligand for transferrin receptor. Transferrin can bind to and mediate the transport of iron [1].
In Vitro, 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 μg/mL in binding buffer (For example, PBS (Phosphate Buffered Saline) or the basic culture medium containing 0.1% BSA). 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).
References:
[1]. González-Maeso J, et al. Agonist-induced desensitization and endocytosis of heterodimeric GABAB receptors in CHO-K1 cells. Eur J Pharmacol. 2003;481(1):15-23.
In Vitro, 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 μg/mL in binding buffer (For example, PBS (Phosphate Buffered Saline) or the basic culture medium containing 0.1% BSA). 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).
References:
[1]. González-Maeso J, et al. Agonist-induced desensitization and endocytosis of heterodimeric GABAB receptors in CHO-K1 cells. Eur J Pharmacol. 2003;481(1):15-23.