Internalization assay

INTERNALIZATION ASSAY OF Tf AND TfR BY  IMMUNOFLUORESCENCE

Transferrin Receptor Internalization

With this assay you can follow the internalization of transferrin receptor (TfR)

• Plate cells on glass coverslips the day before the internalization assay (in order to obtain 50-70% confluency the day of the experiment).
• To perform the TfR internalization assay, incubate cells with the anti-TfR1 antibody (DF1513; mouse, Thermo Scientific; 1:50 dilution in cold medium) for 1 hour on ice in order to inhibit endocytosis.
• Remove the medium containing the antibody.
• One sample (time 0) is washed in cold PBS (1× phosphate-buffered saline) and immediately fixed in paraformaldehyde (see below) after the incubation on ice with the antibody. 
• Perform a release at 37°C in prewarmed medium (not containing the antibody) for the desired timepoints. 
• After desired timepoints of internalization, wash cells in cold PBS on ice.
• Optional: to remove the residual plasma membrane TfR signal, in order to visualize only internalized receptor, it is possible to perform an acidic treatment  (acid wash: 100 mM Glycine-HCl, pH 2.2) to detach the antibody from the TfR not internalized. To visualize the signal from the PM, skip this step. Perform 3 cycles of acid wash:
  • Incubate cells with acid wash for 45 seconds on ice.
  • Remove acid wash.
  • After the last cycle, wash with cold PBS.
• At the end of treatment, fix cells in 4% paraformaldehyde (in PBS) for 10 min at room temperature and wash 3 times with PBS.  

Process fixed cells for fluorescence microscopy:

• Permeabilize cells in  0.1% Triton X-100, 1% bovine serum albumin (BSA)/PBS for 8 min at room temperature or alternatively in 0.1% saponin supplemented with BSA 1% for 30 min at room temperature (with saponin permeabilization remember to keep 0,1% saponin throughout the protocol).
• To prevent non-specific binding of the antibodies, incubate cells with blocking solution (1% BSA/PBS) for 30 min at RT.

Optional: to perform staining with another primary antibody, process fixed cells as follows:

• Incubate cells for 1 hour with the desired primary antibody.
• Wash 3 times with 1X PBS.

• Incubate cells for 30 minutes with fluorescently-labeled secondary antibody (anti-mouse 488-Alexa, Invitrogen, 1:200 dilution) to reveal TfR antibody, and eventually with other secondary antibodies for the additional stainings performed.
• Wash 3 times with 1X PBS.
• Stain nuclei with DAPI for 5 minutes at room temperature.
• Wash again 3 times with 1X PBS.
• Post-fix samples in 4% paraformaldehyde (in PBS) for 5 minutes.
• Mount coverslips with glycerol mounting media.
• Acquire images with confocal microscope. 

Transferrin Internalization

With this assay you can follow the internalization of transferrin ligand (Tf)

• Plate cells on glass coverslips the day before the internalization assay (in order to obtain 50-70% confluency the day of the experiment).
• Incubate cells with Tf-Alexa (Transferrin-Alexa Fluor 555, Invitrogen, cat. n. T35352, 1:100 dilution) in prewarmed medium for the desired timepoints at 37°C. 
• Wash cells in cold PBS.
• Optional: to remove the residual plasma membrane signal, in order to visualize only internalized ligand, it is possible to perform an acidic treatment (acid wash: 100 mM Glycine-HCl, pH 2.2) to detach the ligand from the TfR exposed to the medium. Perform 3 cycles of acid wash:
  • Incubate cells with acid wash for 45 seconds on ice.
  • Remove acid wash.
  • After the last cycle, wash with cold PBS.
• At the end of treatment, fix cells in 4% paraformaldehyde (in 1× PBS) for 10 min at room temperature and washed with 1× PBS. 

Optional: to perform staining with antibodies, process fixed cells as follows:

• Permeabilize cells in 0.1% Triton X-100, 1% bovine serum albumin (BSA)/PBS for 8 min at room temperature or alternatively in 0.1% saponin supplemented with BSA 1% for 30 min at room temperature (with saponin permeabilization remember to keep 0,1% saponin throughout the protocol).
• To prevent non-specific binding of the antibodies, incubate cells with blocking solution (1% BSA/PBS) for 30 min at RT.
• Incubate cells for 1 hour with the desired primary antibody.
• Wash 3 times with 1X PBS.
• Incubate cells for 30 minutes with fluorescently-labeled secondary antibody.
• Wash 3 times with 1X PBS.

• Stain nuclei with DAPI for 5 minutes at room temperature.
• Wash again 3 times with 1X PBS.
• Mount coverslips with glycerol mounting media.
• Acquire images with confocal microscope.