Endocytosis assay

This protocol has been adapted to measure YFP-HA-Orai1 internalization over time in CHO cells.

Material:

• TRvb1 CHO cell line
• Plasmid pDS-YFP-HA-Orai1 encoding a dually tagged Orai1 with YFP at the N-terminus and an HA tag inserted in the second extracellular loop.
• Cy3- and Cy5-conjugated anti-mouse secondary antibodies from Invitrogen. 
• Purified monoclonal anti-HA.11 Epitope Tag antibody (#MMS-101P) from Covance.
• Poly-D-lysine coated glass-bottomed plates (MatTek Corporation).
• Lipofectamine 2000 (Invitrogen).
• Phosphate-buffered saline (PBS).
• Paraformaldehyde, 4% in PBS.
• 0.1% triton.

Protocol:

Cell Preparation and Transfection:

1. For transient transfections, cells were seeded on poly-D-lysine coated glass-bottomed plates (MatTek Corporation) overnight to 50-60% confluency to initiate the experiment on the following day.
2. On the next day, transfect cells with 1.5µg pDS-YFP-HA-Orai1 plasmid DNA using Lipofectamine 2000 (Invitrogen).
3. After overnight incubation, replace transfection mixture with normal medium and incubate cells for another 18-20 hours.

Labelling and Imaging:

1. Remove media from the transfected cells and incubate with monoclonal anti-HA.11 Epitope Tag antibody (1:100 dilution in PBS) at 37°C.
2. At various times (2 min, 4 min, 6 min, 10 min, 20 min) the PBS was removed, and cells were immediately fixed for 10 min in 4% PFA at room temperature |(RT).
3. PFA was then removed, cells washed with PBS, and then stained with Cy5-conjugated anti-mouse secondary antibody (1:400 dilution in PBS with 5% FBS) for 30 minutes at 37°C.
4. The secondary antibody was removed, cells washed with PBS, re-fixed with 4% PFA for 10 min at RT and then washed with PBS.
5. Cells were permeabilized using 0.1 % triton for 10 min at RT.
6. Cells were washed with PBS and then stained with Cy3-conjugated anti-mouse secondary antibody (1:400 dilution) for 30 minutes at 37°C before washing with PBS.
7. The fluorescence intensities of Cy3 and Cy5 were measured using quantitative fluorescence microscopy. The Cy5 signals shows surface Orai1, whereas the Cy3 signal measures accumulation over time of intracellular Orai1 pool bound to the anti-HA antibody.

The (Cy3/YFP)Internal/(Cy5/YFP)Surface ratio is plotted over time and fitted with a linear regression function.