Hello, I am interested in determining whether the binding of our antibody to a cell membrane receptor causes the receptor to be internalized or not. We have data that suggests the antibody prevents receptor cleavage, but this could be due to the antibody preventing proper cleavage or simply causing internalization.
My initial idea was to conjugate our antibody with a fluorescent label (Fluorescein), incubate the antibody with our cells (3T3s), wash the cells to remove unbound antibody, and image the live cells immediately using confocal microscopy to see if the conjugated antibody was internalized.
I have limited imaging experience with fixed cells, but would this idea work? Should we consider doing live cell imaging instead? If so, would the process (incubate, wash, image) be the same?
I would appreciate any help.
Adam Richardson
Good idea!
I would try both, fixed and live cell. However, live cell gives you a more comprehensive dataset as you can easily follow the kinetics of internalization i.e.
If you need a detailed protocol for live cell imaging confocal microscopy analysis, I can send you the following methodological book chapter:
Live cell imaging confocal microscopy analysis of HBV Myr-PreS1 peptide binding and uptake in NTCP-GFP expressing HepG2 cells
DOI: 10.1007/978-1-4939-6700-1_3
In principle it could work. I have some experience looking at the internalization of transferrin receptor using a pulse-chase approach. We did this by loading the receptors with fluorescent transferrin. The cells were kept on ice during loading of the cargo on the cell surface so no internalization took place. Then the coverslips were transferred to 37 degrees to precisely coordinate the start of internalization. You could maybe adapt it to your application.
J Cell Biol. 2002 Mar 4;156(5):797-804. Epub 2002 Mar 4.
Transferrin receptor recycling in the absence of perinuclear recycling endosomes.
Sheff D1, Pelletier L, O'Connell CB, Warren G, Mellman I.
I would like to give an update on what I decided to do. We used a pH sensitive dye conjugated to our antibody of interest that excites at ~5.5 pH as a fluorescent readout for endocytosis with great success. Thank you for your comments.
Hi Adam - I was curious what sort of timing you did for capturing live images, how long you imaged for or did you stick with fixed cells? I wasn't sure how long the antibody stays attached to the receptor or if you could eventually detect recycling? Thanks!
Hi Deanna, we are still working on optimization for our imaging, but have had moderate success with incubating the live cells for 3 hours at 37C in the antibody solution before imaging. We did not do a time course for this study, so imaging only takes ~15 minutes for a 96-well. Also, we have a degradation control that uses a quench-based dye that fluoresces when the receptor is degraded- this might be best for you if you are concerned about degradation/recycling over time.
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