I am planning to perform a degranulation assay using CD107a antibody. Some resources suggest to use inhibitors like monensin to inhibit the intracellular traficking and acidification of the granules which might lead to quenced fluorescence. Though, i come across some suggestions that it is not necessary and in some cases( e.g. if studying function of NK cells) not recommended. I wonder if it is really applicable to perform the experiment by just co-incubation of the stimulated immune cells and CD107a antibody?
Hi Didem,
It is my understanding that to measure CD107a effectively, you must add your actual x-CD107a FACS Ab to the co-culture between NK and target cells. I also routinely add Monensin to be able to simultaneously detect cytokine production by ICS. Hope this helps.
Hi Adam M. Farkas ,
Thanks for the advice! I have another question; how do you prevent non-specific binding of CD107a antibody when you add it directly to the co-culture between NK and target cells?
I co-cultured my target and effector cells to optimize my protocol. However, I observed the same profile of staining in both NK cells which are co-cultured with the target and the NK cells only (no stimulation with any target cell). Any recommendation?
We use monansin in NK cells degranulation assay. It works well.
Let me tell you that we don't co-culture PBMCs but we stimulate PBMCs with PMA+Ionomycin.
I read many papers where cells were imcubatted with K562 cells. In those papers also they treat cells with monansin.
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