if you stimulate NK cells with PMA/I you overpass all the plasma cell events required for NK cell activation. This could be a problem if later on you would like to compare two different populations. PMA/I induces Fasligand in lymphocytes, this could kill other lymphocytes i.e.NK cell could kill themselves. You can avoid it with an antiFasligand in the culture. Finally, for NK cell stimulatin you can use cytokines i.e. IL-2, IL-15, IL-18... or target cells such as K562. I give you the reference of a paper with long-term stimulations:

Human NK cells activated by EBV+ lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells. Diego Sánchez-Martínez, Gemma Azaceta, Aura Muntasell, Nacho Aguiló, David Núñez, Eva Mª Gálvez, Javier Naval, Alberto Anel, Luis Palomera, Carlos Vilches, Isabel Marzo, Martín Villalba and Julián Pardo. Oncoimmunology. 2015. DOI: 10.4161/2162402X.2014.991613

Overstimulation with PMA tends to produce apoptosis. In my case I was using 2ng/ml of PMA in lymphocytes with the same concentration of Iono that you use, and 24h of stimulation was enough to get high levels or apoptotic cells. Are you sure your concentration is not still too high? Besides that, I don't know about NKC but in lymphocytes, the treatment with PMA causes internalization of CD4 (I had to change to intracellular staining for FACS),  maybe there's something similar happening to your cells?

As another massive stimulation method it would be to use PHA, but be careful because is stronger than PMA.

Try to stimulate for 4 to 6 hrs. That worked for me. Even for T cells I've always stimulated for 4-6hrs. I'm assuming that you use golgistop for intracellular cytokines. 

Thanks a lot Jennifer and Sudipt, 

I am stimulating NKC for 3-4 hours, but I will try to minimize the concentration once more. In our case we are looking for NCR & KIR receptors and NK cytotoxicity by measuring CD107a .

How much the percentages of the apoplectic PBMCs is it acceptable after the treatment ?

if you stimulate NK cells with PMA/I you overpass all the plasma cell events required for NK cell activation. This could be a problem if later on you would like to compare two different populations. PMA/I induces Fasligand in lymphocytes, this could kill other lymphocytes i.e.NK cell could kill themselves. You can avoid it with an antiFasligand in the culture. Finally, for NK cell stimulatin you can use cytokines i.e. IL-2, IL-15, IL-18... or target cells such as K562. I give you the reference of a paper with long-term stimulations:

Human NK cells activated by EBV+ lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells. Diego Sánchez-Martínez, Gemma Azaceta, Aura Muntasell, Nacho Aguiló, David Núñez, Eva Mª Gálvez, Javier Naval, Alberto Anel, Luis Palomera, Carlos Vilches, Isabel Marzo, Martín Villalba and Julián Pardo. Oncoimmunology. 2015. DOI: 10.4161/2162402X.2014.991613

Thanks a lot Martin for your valuable comment and the reference.

Normally we stimulate our PBMCs for 4 hours. Is necessary to add Berefeldin A or Menosin to my culture when I am measuring NK cytotoxicity by measuring the expression of CD107a ? And if your answer is yes how much is the optimum concentration I should use to avoid more apoptosis of NK cells ? 

I think we use golgi stop at the concentration recomended by the seller. In 4h it is not toxic, however, do not forget to add the anti-CD107a before the cytotoxicty assay!!

Thanks Martin but why do we use golgi stop for measuring cytotoxicity with CD107a ? 

Does it matter if we do not add golgi stop ? 

I have an additional observation, when you stimulate NK cells  with PMA/ION, CD16 expression drastically reduce even in the lower concentration for 5hrs.

this has already been published one or two years ago. I am not sure if the mechanism is known.

I have reported this  in my publication last year as well

Antiretroviral therapy partly reverses the systemic and mucosal distribution of NK cell subsets that is altered by SIVmac₂₅₁ infection of macaques.(http://www.ncbi.nlm.nih.gov/pubmed/24503100)