Profiling a Killer at the Scene of the Crime:

The Natural Killer Cell Cytotoxicity Assay

Natural killer cells (NK cells) are a subset of blood lymphocytes that play an essential role in immunosurveillance and the innate immune response.   As a first line of defense, NK cells specifically recognize virally infected and malignantly transformed cells and target them for killing and removal.   And they work quickly:  in contrast to T cells, NK cells can distinguish between healthy and abnormal cells in the absence of the MHC (Major Histocompatibility Complex), which allows for rapid and efficient killing.   When NK cells encounter a target, they release the contents of their cytotoxic granules (perforin, granzymes) into the immune synapse.  Also, upon interaction with a target cell, NK cells secret the cytokines interferon gamma (INFγ) and Tumor Necrosis Factor-α (TNFα).   NK cytotoxic function varies among individuals:  decreased NK cell function has been observed in cancer patients and in patients with recurrent viral infections.  Thus the measure of NK cell effector function (or cytotoxicity) has become an important assessment in many disease states. 

Past and Present Assays to Measure NK Cytolytic Activity

Since 1968, the “gold standard” for measuring the cytolytic activity of NK cells was the chromium release assay (CRA).  In the CRA, target cells were radiolabel with Chromium (51Cr) and incubated with effector cells at varying ratios for 4-16 hours.  51Cr released from lysed target cells into the supernatant was then quantified using a gamma counter.  Although used for many years, there were a number of drawbacks to the CRA:  the assay involves the use, handling, training and disposal of radioactivity.  Also, there was increased background in the assay due to the spontaneous release of 51Cr from the target cells, and high inter-assay and Inter-lab variability, all of which made the CRA not suitable for clinical lab standardization.

Because of these drawbacks, non-radioactive, stream-lined, flow-cytometry based NK cytotoxicity assays were developed that were faster, less expensive, and more amenable to standardization.  Some variations of the flow-based assays exist, but the basic format  involves  co-incubating  peripheral blood mononuclear cells (PBMC) (or purified NK cell preparations) at different ratios with a target tumor cells  that are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells within the PBMC).  After the incubation period, killed target cells are identified by a nucleic acid stain, which permeates dead cells.

When  the CRA and flow-based Cytotoxicity assays were compared head to head using the same donor PBMCs, the correlation between the assay results was high (r=0.85, Kane, et al,  1996;   r=0.90-0.99, Kim et al.,  2007;  r=0.89, PBMC and  purified NK cells (r=0.89), Jang e et al.,  2012).  The flow-based method is amenable to both diagnostic and research applications and, because of the powerful capabilities of multi-parameter flow cytometry, has the added advantage of deeper analysis of NK cell phenotype and function.   For example, in one variation of the assay, Tognarelli et al., 2016,  describes the basic flow-based NK cytotoxicity assay (using magnetic bead-purified NK cells), with added endpoints of NK profiling (CD56+CD16+ subtypes), function (CD107a, a surface marker of degranulation), and intra-cellular cytokine staining (INFγ and MIP1β) within a single assay.

At Flow Contract Site Laboratory (FCSL), we have developed and offer a flow cytometry based NK cytotoxicity assay to evaluate human and non-human primate NK cell effector function.     K562 target cells are labeled with PKH67 (a fluorescent green dye that labels the cell membrane) to discriminate target cells from effector cells.  Then, effector cells (PBMCs) are incubated with the PKH67 labeled K562 target cells at different Effector to Target (E:T) cell ratios from 100:1,   50:1 and 25:1  (Figure 1).

Figure 1. Schematic representation of different ratios of Effector to Target cells. After co-culture for 4 hours at 37°C, 5% CO2, the cell mixtures are stained with 7-AAD (7-Aminoactinomycin D) which is a fluorescent chemical compound with a strong affinity for DNA used to detect dead cells (Figure 2).

Figure 2. Schematic representation of the interaction of PKH67 labeled K562 target cells with NK effector cells in PBMC.

The percentages of 7AAD positive (dead) K562 target cells are determined by flow cytometry at each Effector to Target (E:T) ratio tested.    Figure 3 shows a comparison of NK cell cytotoxicity in human and Non-Human Primate (NHP) in the presence or absence of IL-2, a cytokine which can enhance NK cell effector function.  In addition to evaluating NK cytolytic activity, each donor can be immunophenotyped for their NK cell number:   NK cells are identified as CD3-CD16+CD56+ in human donors and CD3-CD16+ in NHP donors.

Figure 3. Ratio of Effector: Target cells (E:T) vs the % Cytotoxicity of human and NHP NK cells. Human and NHP NK cytolytic activity is highest at the 100:1 E:T ratio, with a dose-dependent decline as the E:T ratio decreases. IL-2 enhances both human and NHP NK cytotoxicity.

Recently, decreased NK cell activity has been implicated in the presentation of Chronic Fatigue Syndrome, with the reduction of NK cell lytic activity in patients with the disease compared to healthy controls (Brenu et al., 2013).  In collaboration with The Centers for Disease Control and Prevention (CDC), FCSL has been using our flow cytometry based NK assay to assess NK function in support of an ongoing clinical trial.  Results from these efforts were presented in a poster entitled “Pilot Study Evaluating Impact of Sample Processing and Assay Format on Measured Natural Killer Cell Function” at the 12th International IACFS/ME Research and Clinical Conference in 2016.  These applications of a NK Cytotoxicy assay illustrate the potential for added value in preclinical or clinical trials where changes in NK activity are expected to impact outcomes.

FCSL is a contract flow lab that provides high throughput and high capacity flow cytometry services, running multiple flow cytometers with up to 10 color antibody panels daily.  We are proficient in processing a multitude of specimen types including whole blood, frozen PBMCs along with cell culture and tissue processing capabilities. Our flexibility in handling so many specimen types allow for the support of a wide range of flow cytometry assays including: immunophenotyping/lymphocyte subset analysis, receptor occupancy, functional assays and cell viability/apoptosis measurements. Our expert staff is always available to help guide you through these tests and we welcome clients to visit our facility. We encourage sponsor engagement throughout the process. Contact us for more information!


  1. W. Brenu, S. L. Hardcastle, G. M. Atkinson, M. L. van Driel, S. Kreijkamp-Kaspers, K. J. Ashton, D. R. Staines, S. M. Marshall-Gradisnik. Natural killer cells in patients with severe chronic fatigue syndrome. Auto Immun Highlights. 2013 Dec; 4(3): 69–80.

Y.Y Jang, D. Cho, S. Kim, D. Shin, M. Park, J. Lee, M. Shin, J. Shin, S. Suh, and D. Ryang.   An Improved Flow Cytometry-Based Natural Killer Cytotoxicity Assay Involving Calcein AM Staining of Effector Cells, Ann Clin Lab Sci Winter.  2012 42: 42-49.

  1. Kandarian, G. M. Sunga, D. Arango-Saenz, M. A. Rossetti. Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity. J. Vis. Exp. 2017 (126), e56191, doi: 10.3791/56191.

K.L Kane, F.A. Ashton, J. L. Schmitz, J.D Folds, Determination of Natural Killer Cell Function by Flow Cytometry,  Clinical and Diagnostic Laboratory Immunology.  1996   3 (3):  295-300.

G.G. Kim, V.S. Donnenberg, A.D. Dennenberg, W Gooding, T.L Whiteside,  A Novel Multi-parametric Flow Cytometry-based Cytotoxicity Assay Simultaneously Immunophenotypes effector cells:  Comparisons to a 4 h 51Cr-release assay, J Immunol Methods. 2007 August 31; 325(1-2): 51–66.

  1. Tognarelli, B. Jacobs, N. Staiger, E. Ulrich, Flow Cytometry-based Assay for the Monitoring of NK Cell Functions, J. Vis. Exp. 2016 (116), e54615, doi:10.3791/54615
  2. Watzl, How to Trigger a Killer: Modulation of Natural Killer Cell Reactivity on Many Levels. Adv Immunol. 2014 124:  137-170.