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Application Note

Identification and Functional Characterization of Natural Killer Cells Using Flow Cytometry
Natural killer (NK) cells are a subset of lymphocytes involved in innate immunity (Figure 1). Their mechanisms of action include the direct lysis of non-self target cells through release of perforin and granzymes, as well as the production of cytokines that are supplied to other innate immune cells, such as monocytes and macrophages. Given the important roles that NK cells play in the immune response, the study of their characteristics and function represents a major field in immunology. Although NK cells are typically present within very heterogeneous immune cell populations, they can be identified by characteristic cell surface markers. NK cells are typically CD3-CD56+ lymphocytes; however, CD3- NK cell subsets include CD56brightCD16 immunoregulatory NK cells, which express high levels of cytokines, and the CD56+CD16+ subset of non-cytokine-expressing but highy cytotoxic NK cells. CD3+CD56+ NK-T cells are a subset of T cells involved in recognition of lipid antigens. NK-T cells, however, possess some properties of NK cells, including cytokine production and cytotoxicity.
Cytokine Production Granule Release (Cytotoxicity) Inhibitory Receptor MHC Class I

Data Sheet


Activation Receptor


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Activation Receptor Ligand



Figure 1. Natural Killer (NK) cells are involved in innate immunity, through lysis of target cells. Cytotoxicity is activated through a combination of a positive signal, mediated by an activation receptor on the target, with the lack of an inhibitory signal, which in this case is the absence of major histocompatibility complex I (MHC I) self molecule (A). Activation of this response results in the release of perforin and granzyme particles, which act to puncture target cell membrane, and trigger apoptosis, respectively (B).

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Flow cytometry is a popular technique for studying NK cells in vitro and in vivo. Simultaneous labeling of lymphocytes with fluorescent antibodies specific for CD16, CD56 and CD3, followed by flow cytometry analysis, enables the identification of NK cells and distinguishes them from NK-T cells. The combination of these three antibodies enables a researcher to identify natural killer cells from a variety of relevant source biomaterials, including human PBMCs and whole blood samples. Here, we describe the use of a novel flow cytometry kit to characterize NK cells in human peripheral blood. In addition, we also describe the use of flow cytometry to perform a cell cytotoxicity test to detect NK-mediated killing of human K562 myeloma cells. By measuring both antibody staining and autofluorescence, we distinguished unbound target cells and cells that have been targeted by NK cells. We monitored cell death by the use of a fixable viability dye, eFluor660. This dye is excited by the red (633nm) laser and is fully compatible with cell processing protocols that require fixation and permeabilization. Because it has an emission pattern that is specific to the Red2 channel, its signal does not interfere with the Green and Yellow channels, a problem commonly associated with propidium iodide staining. The resulting multiparameter analysis enables the measurement of viability of specific cell subpopulations within a heterogeneous sample.

After completion of lysis, samples were centrifuged at 400 x g for 5 minutes and the supernatant was removed. Cell pellets were washed with 250 L of 1x Assay Buffer to remove unbound antibodies and centrifuged at 400 x g for 5 min. Supernatant was discarded and wash step repeated. Pellets were resuspended in 250 L of 1x Assay Buffer prior to analysis. Cell cytotoxicity testing Negatively selected human NK cells were stained with anti-CD56 PE, and then incubated with K562 human myeloma cells at log phase at an effector:target ratio of 5:1. Following incubation, PE fluorescence combined with green autofluorescence enabled identification of unbound K562 cells and K562 cells that were being targeted by NK cells. The eFluor 660 fixable viability dye was then utilized to identify live cells in each population.

We assessed the ability to quantitate lymphocyte subsets from fresh, human whole blood (Figure 2). By first applying an elliptical gate to the side scatter v. forward scatter dot plot, we identified the lymphoid population. We applied this gate to two dot plots, anti-CD56-PE v. anti-CD16-FITC and anti-CD56 PE v. anti-CD3 APC. From the resulting statistics, we determined that approximately 12% of the population were CD3-CD56+ NK cells and12% of the cells were CD56+ CD16+ NK cells.

Materials and Methods

All cells were analyzed by flow cytometry, using the FlowCellect human NK cell characterization kit, which can be used to stain NK cells from a variety of tissue types, including PBMCs and whole blood. Flow cytometry was conducted using a guava easyCyte benchtop flow cytometry system and the InCyte data acquisition and analysis software package. At least 100,000 events were acquired per sample. Analysis of NK cell subpopulations in whole blood Fresh, human whole blood (10 L) was added to each well of a 96-well plates. 5 L each of anti-CD3 APC, anti-CD16 FITC, and anti-CD56 PE were added to the blood in each well. The wells were mixed gently and incubated in the dark at room temperature (RT) for 20 min. After Fixation Solution (1:40) was added to each well, red blood cells were lysed by adding 180 L of Guava 1x Lysing Solution to each well. Wells were mixed by pipetting and incubated at RT in the dark for 30 min.
4.7% 12.3%







Figure 2. Identification of lymphocyte subsets from whole blood. Fresh human whole blood was stained with anti-CD3 APC, anti-CD16 FITC, and anti-CD56 PE in accordance with the FlowCellect Human NK cell characterization kit user guide. Lymphoid events were further gated for CD16 and CD56 or CD3 and CD56.

Next, we used the NK cell characterization kit to assess the proportion of K562 target cells that were bound to (and being killed by) effector NK cells. In a 5:1 effector cell:target cell population, 8% of the K562 cells were bound to NK cells (Figure 3B). 84% of the bound K562 cells were viable (Figure 3C) stained with fixable viability dye), while 96% of the unbound K562 cells were viable (Figure 3D).
A. B.

Natural Killer cells (CD3-CD16+CD56+) are a major players in innate immunity, both as direct cytotoxic effectors as well as regulators for other innate immunity cell types. We have shown that, using the FlowCellect human NK cell characterization kit, one can achieve accurate phenotyping on a variety of sample types, including whole blood samples. Using the same kit to perform an NK cell cytotoxicity test, we demonstrate that unbound K562 target cells can be clearly distinguished from those that have been engaged by CD56+ NK cells, and each of these populations can be further investigated for viability using the eFluor 660 dye.


162 (8%)


1793 (92%)

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FlowCellect Human Natural Killer Cell Characterization Kit

4.4% dead

FCIM025164 0500-4008

16.0% dead

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Viability (Bound K562s)

Viability (Unbound K562s)

Figure 3. Cytotoxicity of NK cells shown by incubation with K562 human myeloma cells in a 5:1 Effector:Target (E:T) ratio. Panel A shows the distinct scatter patterns between effector and target cells, which enables one to distinguish between the two cell types. Panel B shows the use of CD56 staining and green autofluorescence to distinguish between effector and target cells, as well as between targets that are bound to NK cells from those that are free. Number of each and their percentages are shown. Panels C and D show the viability staining of bound and unbound K562 populations, respectively, with the percentage of dead events in each case.

FCIM025123 FCIM025124 FCIM025125 FCIM025137 FCIM025138

FCIM025118 FCIM100158

FCIM025126 FCIM025168

1. Cooper, MA et al. The biology of human natural killer-cell subsets. Trends in Immunology. 2001; Vol.22 No. 11: 633-640. 2. Ren, F et al. Assays of Natural Killer (NK) Cell Ligation to Target Cells. Current Protocols in Cytometry. 1998; 9.10.1-9.10.8. 3. Zamai, L et al. Kinetics of In Vitro Natural Killer Activity Against K562 Cells as Detected by Flow Cytometry. Cytometry. 1998; Vol 32: 280-285.

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FlowCellect, guava easyCyte, InCyte, EMD Millipore and the M mark are trademarks of Merck KGaA, Darmstadt, Germany. Lit No. AN4422EN00 LS-SBU-12-06398 05/2012 Printed in the USA. 2012 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.