There are some options in the literature, pre-culture with K562 cells, add IL-2 or IL-15 or antibodies against NKG2D or NKp46... But I don't know which one would be the best and fastest way to do it. Does anybody have experience with this?
It all depends on your question.
priming cells with exogenous cytokines will renders your cells more efficient for cytokine and perforine release without decreasing activating receptor expression. using cells may exhaust your cells and reduce surface expression of activating receptors. for low pre-activation we use IL-2 (100Ui/ml)+IL-15 (10ng/ml) overnight. for a stronger stimulate your cells with IL-2 1000UI/ML or scale up the cocktail IL-15+2
hope it helps
Cyril
Hi Maria
It does depend on exactly what you're looking for as different stimulators activate different pathways and it depends on whether you want to grow an NK line or not. If your primary goal is just to activate primary NK cells from peripheral blood and look at killing activity asap, then we use IFN-alpha. The IFNa alone (eg. at 500 iu/ml) activates NK cells within 6 hours (though we normally do this overnight as standard) and seems to have less effects on the expression of NK receptors. If you want to 'superstimulate' the NK cells, then this can be achieved with further addition of IL-15, which will upregulate a range of NK activating receptors.
Best regards
Ed
It all depends on your question.
priming cells with exogenous cytokines will renders your cells more efficient for cytokine and perforine release without decreasing activating receptor expression. using cells may exhaust your cells and reduce surface expression of activating receptors. for low pre-activation we use IL-2 (100Ui/ml)+IL-15 (10ng/ml) overnight. for a stronger stimulate your cells with IL-2 1000UI/ML or scale up the cocktail IL-15+2
hope it helps
Cyril
Ibrahim Abdalla · 2.36 · 1.46 · University of Leeds
As Dr. Eddie mentioned above, it depends on what you intend to use theses activated NK cells for and whether they are primary NK cells from PBMCs or NK cell line like NK92, i.e. assessing their killing ability, IFN-g production..... you have several option including PMA/ionomycin from( 50ng/ml& 500ng/ml) from sigma , IL-2 (50U/ml), IL-15 (20ng/ml)from R&D, and also IL12/IL-18 (10ng/ml &100ng/ml) from R&D and MBL; in addition tp IFN-g and IFN-a these are some protocols that we regularly use in our lab and they are proved effective, if stored and added properly.
Just to add that it would take about a week for primary NK cells to be fully activated in case of IL-2 and IL-5. and if you add excessive activation your render your cells exposed to activation induced cell death (AICD) which you would see on the FCS/SSC plot if you are using flowcytometry.
Bet of luck
Thank you
We finally used IL-2 plus IL-15 overnight and works fine to pre-activate NK cells from PBMCs to assess their killing ability. Thanks to everyone for the kind answers!
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