Hello! I rarely worked with ADCC-assays, but in different protocols I met a mentions of different cells types:
1) PBMC's (ficoll-purified, without any further separation)
2) NK-cells, purified from PBMC's by cell sorter (with this kind of assays I worked)
3) NK-92
4) THP-1 (PMA transformed, for ADCP)
What differences in results should I expect by changing cells mentioned in protocol, by cells available in my lab? (e.g. change NK-92 by donor's NK-cells)
Hello Dmitry Lifanov
ADCC is a result of Fc-gamma receptor (FcγR) mediated interaction with effector immune cells such as natural killer (NK) cells, macrophages and granulocytes. The binding of FcγR to the Fc domain induces the release of both granzyme and perforin from effector cells, leading to target cell lysis and Fc-dependent tumor cell phagocytosis.
I would recommend the use Natural Killer cells from human donors which is FcγRIIIa +ve or FcγRIIIa engineered NK-92 cells.
Macrophages in varying states of activation differ in their ability to perform ADCC. Moreover, by using PBMCs there is a probability of an increased inter assay variability.
So, in order to avoid variability which is caused by differences in immune status and genetic background of donor cells use of NK-92 cells would be preferable.
Best.
You will want to stain for cd16, the key fc receptors for adcc in nk cells. I believe that the parental nk-92 cell line is deficient in cd16 and therefore adcc may be problematic using the parental cell line. It was later engineered to express it...hence my suggestion that you stain to confirm. Human pbmc nk cells are cd3neg cd56 pos but variable cd16 pos and different donors will have different frequency of nk cells in the pbmc and different frequencies if cd16pos nk cells.