Hi,
I am interested in cells that express a specific receptor. I use a fluorophore-conjugated antibody bound to the receptor followed by MACS positive selection to enrich for this receptor-expressing population. After this selection, I culture the cells for 5-7 days and would like to test functional activity of the receptor used for positive selection. Would the antibody used for selection stay bound to this receptor (for how long), and is there a way to dissociate it in a way that would still allow for re-culturing of the cells after?
Thank you,
Vinita Joshi
Most likely, the beads would be internalized by endocytosis and destroyed in lysosomes. The receptor protein would be re-made, just like it is all the time during normal turnover. I think this process is likely complete in 5-7 days. But every protein is different. So you should test it.
The acidic environment of the lysosome would literally destroy the magnetic particle itself. So you could test when the cells stop being magnetic. If they're not magnetic at all anymore, then I think you could view them as fully renewed for your functional assay. I bet your culture period is long enough.
Receptors usually have a pretty quick turnover. I'd expect all of the fluorescent antibody be gone after a few hours. So you could re-examine your cells with a fluorescence microscope after various time points to get an idea about the speed of the process.
If that should fail, try treating your cells with trypsin or another suitable protease.
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