Both cytotoxicity and degranulation assays can easily be performed using a four-color flow cytometer. For cytotoxicity, target cells can be pre-stained with CFSE. After incubation with NK cells at different E/T ratios, cells are stained with a live/dead marker. You can use traditional dyes like PI or 7-AAD, but we normally use LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit from Invitrogen in the “APC” (aka FL-4) channel.

In theory, degranulation and cytotoxicity can be determined in the same well using a PE-labeled antibody to CD107. However, cytotoxicity is more effective at higher E/T ratios, while degranulation responses are stronger at low E/T ratios.

Natural cytotoxicity can be assessed using the cell lines indicated above – K562 cells are readily killed by resting NK cells (i.e. not cytokine-stimulated). For other cell lines, pre-stimulation by IL-2, IL-12 or IL-15 may be needed to observe robust cytotoxicity responses.

By combining anti-CD20 antibodies (e.g. Rituximab) and CD20-expressing target cells (e.g. 721.221 or Daudi cells) you can also study NK cell-mediated ADCC.

K562 is used because lack of MHC expression makes NK cells particularly reactive and the assay works very well. Don't have specific protocols.

I have experience with epithelial cell lines. 4 h NK-mediated killing after proliferation and activation (3 days with stimulus and epithelial cells) is working in my hands.

Good luck.

Hi Milad,

the standard protocole to assess degranulation is to use K562 or 721.221 cell line and to mesure by flow cytometry the degranulation marker CD107a. For the direct cytoxicity assay, do you have the authorization in your lab to use radioactif material ?

Thank you for your answer dear Olivier,

Not actually! No radioactive materia; however, we have four color flow cytometry and it would be great if we could do all the tests using flow cytometry-based methods..

Sure so CD107a is the best way. Are you working with bulk PBMC or purified NK cell ?

Yes, I guess CD107a would be the best. I am about to use purified NK (Miltenyi biotech MACS kit)..

Is it possible for u to send me the detailed protocols that you use in private?

Both cytotoxicity and degranulation assays can easily be performed using a four-color flow cytometer. For cytotoxicity, target cells can be pre-stained with CFSE. After incubation with NK cells at different E/T ratios, cells are stained with a live/dead marker. You can use traditional dyes like PI or 7-AAD, but we normally use LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit from Invitrogen in the “APC” (aka FL-4) channel.

In theory, degranulation and cytotoxicity can be determined in the same well using a PE-labeled antibody to CD107. However, cytotoxicity is more effective at higher E/T ratios, while degranulation responses are stronger at low E/T ratios.

Natural cytotoxicity can be assessed using the cell lines indicated above – K562 cells are readily killed by resting NK cells (i.e. not cytokine-stimulated). For other cell lines, pre-stimulation by IL-2, IL-12 or IL-15 may be needed to observe robust cytotoxicity responses.

By combining anti-CD20 antibodies (e.g. Rituximab) and CD20-expressing target cells (e.g. 721.221 or Daudi cells) you can also study NK cell-mediated ADCC.

Thank you for your detailed answer dear Dr. Thorén. Quite helpful

To estimate the degranulation of NK cells; NK cells were cultured with target K562 cells at effector to target ratios (1:1, 1: 2) in U bottom 96-wells plate then added anti-CD107a mAb conjugated with fluorchrome (Fitc) in the presence of GolgiStop to prevent CD107a degradation. Stimulation could be for 3- 6 hours. After the end of incubation samples were stained with surface markers such as CD56 (PE), CD3 (Percp) and/or CD16(APC and measured by Flow Cytometry.

Hi Milad,

I totally agree with Fredrik and Ameera protocol. I will just add that before putting the golgi Stop, to incubate during one hour the cells with the CD107a at 37 degrees . It will give you a better signal. Then I usually put golgi Stop during 4 hours. Good luck

Thank you all for your valuable comments.

Regards,

Milad