Hi!
I´m trying to set up a protocol for the evaluation of the internalization of a radiolabelled antibody; all the protocols that I´ve found suggest to lysate the cells and then to measure the internalized fraction of an antibody.
Is this a fundamental step? Or it´s just possible to wash the cells, collect them and measure the radioactivity with the gamma-counter?
Thank you!
Cheers
Hi Stefania,
We incubate cells at 37°C with radiolabeled antibody and at each time point separate supernatant, cell-bound fraction and cells (internalized fraction) for gamma-counting. Article Quantitative ImmunoPET of Prostate Cancer Xenografts with Zr...
Thank you for your answer!
Is it strictly necessary to destroy the cell membrane before measuring the internalized antibody?
And You keep the cells in the incubator without shaking for all the time points?
Yes, these are adherent cells that we incubated without shaking.
We used a 'stripping buffer' to remove bound but not internalized antibody from the cell surface. That should NOT disrupt the cell membrane, otherwise you would mix the cell bound fraction with the internalized fraction.
Ok, so why do you use this buffer: PBS, 10mM Tris, 0.5% SDS, pH 7.4 for the internalized fraction? Could I also use only PBS?
Thank you again!
We performed the internalization assay on adherent cells, so after stripping the membrane bound fraction of the cells, we lyse the cells to harvest the internalized fraction. If your cells are already in suspension or if you prefer using a cell scraper, the buffer won't make a difference.
Kirstin A Zettlitz Hi, Kirstin, would you please share your protocol specifically with me for the internalization assay? Recently I want to perform a Internalization Assay but I can't find the very nice protocol for this, Thank you very much.
Hi Fusheng, you can find the method in the supplementary information of this paper:
Article Quantitative ImmunoPET of Prostate Cancer Xenografts with Zr...
Kirstin A Zettlitz I´ve another question for you: did you eve checked wheter the cells treated with the buffer still have an entire membrane afterward?
Kirstin A Zettlitz Hi kirstin, thank your very useful document for the internalization assay, i want to perform a FACS based Internalization assay and made a protocol in detain based on your shared paper, would you mind give me some suggestion if there are something wrong in some stpes (please return the edited version if it is posible).
Thank you for your help!
Stefania Pezzana. The cell membrane is intact after stripping the cell surface, otherwise we would have no cells left. However, some cell lines might be more sensitive than others. If you are worried that your cells might lyse from that treatment, you could also skip that step and just look over time if the radioactivity associated with the cells saturates quickly (that would be cell surface bound) or increases (by internalization) over time.
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