We would like to thank Jill Narciso, UCLA Immunogenetics Center, for her assistance with manuscript preparation.

Samples were collected according to the ethical guidelines established by the UCLA Human Research Protection Program and IRB approved.
1. Preparation of reagents
NOTE: Unless otherwise stated, all reagents should be allowed to equilibrate at room temperature prior to use. All reagents must remain sterile.
<ol>
	<li>Prepare a 2x working solution of Tween-20 (i.e., 0.2%) by adding 10&#160;&#181;L of Tween-20 solution to 5 mL of phosphate-buffered saline (PBS) withou...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+natural+killer+cell+function+by+flow+cytometry.">Determination of natural killer cell function by flow cytometry.</a> Clin Diagn Lab Immunol. 3 (3), 295-300 (1996).</li><li>Jang, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+flow+cytometry-based+natural+killer+cytotoxicity+assay+involving+calcein+AM+staining+of+effector+cells.">An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells.</a> Ann Clin Lab Sci. 42 (1), 42-49 (2012).</li><li>Korzeniewski, C., Callewaert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-release+assay+for+natural+cytotoxicity.">An enzyme-release assay for natural cytotoxicity.</a> J Immunol Methods. 64 (3), 313-320 (1983).</li><li>Karimi, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+cytotoxicity+by+bioluminescence+imaging+outperforms+the+standard+chromium-51+release+assay.">Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.</a> PLoS One. 9 (2), e89357 (2014).</li><li>Oppenheim, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyco-engineered+anti-EGFR+mAb+elicits+ADCC+by+NK+cells+from+colorectal+cancer+patients+irrespective+of+chemotherapy.">Glyco-engineered anti-EGFR mAb elicits ADCC by NK cells from colorectal cancer patients irrespective of chemotherapy.</a> Br J Cancer. 110 (5), 1221-1227 (2014).</li><li>West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., Herberman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+cytotoxic+reactivity+of+human+lymphocytes+against+a+myeloid+cell+line:+characterization+of+effector+cells.">Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.</a> J Immunol. 118 (1), 355-361 (1977).</li><li>Jedema, I., van der Werff, N. M., Barge, R. M., Willemze, R., Falkenburg, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+CFSE-based+assay+to+determine+susceptibility+to+lysis+by+cytotoxic+T+cells+of+leukemic+precursor+cells+within+a+heterogeneous+target+cell+population.">New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population.</a> Blood. 103 (7), 2677-2682 (2004).</li><li>Lecoeur, H., Fevrier, M., Garcia, S., Riviere, Y., Gougeon, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometric+assay+for+quantitation+and+multiparametric+characterization+of+cell-mediated+cytotoxicity.">A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.</a> J Immunol Methods. 253 (1-2), 177-187 (2001).</li><li>Carotta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+NK+Cells+for+Anticancer+Immunotherapy:+Clinical+and+Preclinical+Approaches.">Targeting NK Cells for Anticancer Immunotherapy: Clinical and Preclinical Approaches.</a> Front Immunol. 7, 152 (2016).</li><li>Mandal, A., Viswanathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cells:+In+health+and+disease.">Natural killer cells: In health and disease.</a> Hematol Oncol Stem Cell Ther. 8 (2), 47-55 (2015).</li><li>Rezvani, K., Rouce, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Application+of+Natural+Killer+Cell+Immunotherapy+for+the+Treatment+of+Cancer.">The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer.</a> Front Immunol. 6, 578 (2015).</li><li>Angelo, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+NK+cell+phenotyping+and+variability+in+healthy+adults.">Practical NK cell phenotyping and variability in healthy adults.</a> Immunol Res. 62 (3), 341-356 (2015).</li><li>Zons, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+europium+and+chromium+release+assays:+cytotoxicity+in+healthy+individuals+and+patients+with+cervical+carcinoma.">Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma.</a> Clin Diagn Lab Immunol. 4 (2), 202-207 (1997).</li><li>Yovel, G., Shakhar, K., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+sex,+menstrual+cycle,+and+oral+contraceptives+on+the+number+and+activity+of+natural+killer+cells.">The effects of sex, menstrual cycle, and oral contraceptives on the number and activity of natural killer cells.</a> Gynecol Oncol. 81 (2), 254-262 (2001).</li><li>Laue, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+NK+cell+function+in+obese+healthy+humans.">Altered NK cell function in obese healthy humans.</a> BMC Obes. 2, 1 (2015).</li><li>Hazeldine, J., Lord, J. 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The authors declare no conflicts of financial interest.

The method described here provides a straightforward and cost-effective alternative to the traditional 51Cr release assay to assess NK cell cytotoxic activity. This method is sensitive, reproducible, and less time-consuming than previous standard methods, like CRA, and can be used for both clinical and research applications.
While the assay works with both total PBMCs and enriched NK cells, the option to use PBMCs without the need to purify cell populations is a great benefit when d...

An erratum was issued for: <a href="https://www.jove.com/video/56191/a-flow-cytometry-based-cytotoxicity-assay-for-assessment-human-nk">A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity</a>. Figure 4 has been corrected to show background-corrected data.
Figure 4 was updated from:
<img alt="Figure 4" src="/files/ftp_upload/56191/56191fig4v1.jpg" />
to:
<img alt="Figure 4" src="/files/ftp_upload/56191/56191fig4v2.jpg" />

An erratum was issued for: <a href="https://www.jove.com/video/56191/a-flow-cytometry-based-cytotoxicity-assay-for-assessment-human-nk">A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity</a>. Figure 4 has been corrected to show background-corrected data.

Erratum: A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Natural killer (NK) cells are a sophisticated subset of human innate lymphocytes critically involved in the elimination of virally infected cells, transformed cells, and other pathogenic threats 1,2. NK cell lytic granules house cytotoxic proteins, such as perforin and granzymes. Upon activation, NK cells form a complex interaction with their targets known as immunological synapse, whereby these cytolytic molecules are locally released, resulting in direct target cell lysis and apoptosis, together with cytokine and chemokine release and ultimately in the induction of an inflammatory state 1,3,4.
NK cell activation involves a complex string of activating and inhibitory interactions between NK cell receptors and ligands expressed on the surface of target cells, forming a tightly regulated system. One of the most studied mechanisms of NK cell activation is the &#34;missing self&#34;. Indeed, lack of detection of class I major histocompatibility complex (MHC), or human leukocyte antigen (HLA) molecules, on infected or transformed cells triggers NK cell cytotoxicity. Tumor and virus-infected cells generally downregulate these antigens to escape T cell-mediated immunity, thus becoming primary NK cell targets 1,3,4.
Assessment of NK cell function is primarily categorized into degranulation or cytotoxicity assays. However, degranulation assays, such as flow cytometric detection of the degranulation-associated marker CD107a, are only indicative of NK cell activation and not of their ultimate function, the direct killing of target cells 5,6,7,8. Hence, this limitation has drawn investigators to cytotoxicity assays as a more telling and more direct alternative.
The long-time &#34;gold standard&#34; for assessing cell-mediated cytotoxic activity of both T and NK cells is the chromium release assay (CRA). CRA involves radioactively labeling of target cells with 51Cr and co-incubating them with effector cells. This assay is steeped in the principle that cell lysis results in the release of protein-bound 51Cr into the supernatant, which can be measured by gamma counting. This assay, while effective, is problematic for a variety of reasons: high material costs, handling and disposal of radioactive 51Cr, spontaneous release of 51Cr, and difficult standardization - making it altogether impractical 9,10.
A number of non-radioactive assays, involving fluorescent labeling, enzyme release, and even bioluminescence, have since been developed as alternatives to CRA 11,12,13,14. We describe here a flow cytometry-based method for measurement of NK cell cytotoxic activity on K562 target cells that is simple, sensitive, and reproducible. K562 cells are a human erythroleukemic cell line with reduced expression of HLA class I and heightened expression of ligands for activatory NK receptors, which makes them particularly susceptible to NK cell-mediated cytotoxicity 15. In this assay, K562 cells are pre-labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and co-cultured at various ratios with either peripheral blood mononuclear cells (PBMCs) or purified NK cells 1. CFSE is a stable, protein-binding fluorescent dye that allows discrimination of target cells from effector NK cells 16,17. After the co-incubation, a nucleic acid stain, specifically permeating the membrane of dead cells, is used to identify killed target cells (see table of materials). The samples are then acquired on a flow cytometer to determine the percentage of dead (i.e., stain+) CFSE+ target cells.
This assay can be used as a routine diagnostic screening for monogenic defects affecting the NK cell compartment, of which there are approximately 30 known defects causing either functional or classic NK cell deficiency, and for primary or secondary hemophagocytic lymphohistiocytosis. It is also useful to investigate NK cell activity in patients with recurrent, severe herpes viral infections, to evaluate immune reconstitution following hematopoietic cell transplantation or post immunomodulatory therapy 18,19,20, and for a host of basic research applications.

<table>
	<tbody>
		<tr>
			<td>Phosphate-buffered Saline (1x, w/o Ca2+ and Mg2+)</td>
			<td>Corning (Cellgro)</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Corning (Cellgro)</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal Bovine Serum</td>
			<td>Omega Scientific</td>
			<td>FB-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-163</td>
			<td>Stock solution at 10,000 U/mL</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL-050</td>
			<td>Lyophilized from a 0.2 &#956;m filtered solution in Acetonitrile and TFA with BSA as a carrier protein. Reconstitute with 500 ul at 100 &#956;g/mL in sterile 100 mM Acetic Acid containing at least 0.1% bovine serum albumin (2.1x10E6 IU/ml)</td>
		</tr>
		<tr>
			<td>K562 Cells</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td>Cancer cell line&#160;</td>
		</tr>
		<tr>
			<td>T-75 cell culture flasks</td>
			<td>Corning</td>
			<td>431464</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE cell proliferation kit</td>
			<td>Life Technologies (CellTrace)</td>
			<td>C34554</td>
			<td>Reconstitute I vial with 18 ul DMSO to prepare a 5mM stock solution. Do not freeze/thaw.</td>
		</tr>
		<tr>
			<td>Sytox Red</td>
			<td>Life Technologies</td>
			<td>S34859</td>
			<td>Stock solution is provided at 5 &#956;M in 1 mL DMSO. The DMSO solution may be subjected to multiple freeze-thaw cycles without reagent degradation.</td>
		</tr>
		<tr>
			<td>Sodium/lithium heparin blood collection tubes</td>
			<td>BD</td>
			<td>02-687-95</td>
			<td></td>
		</tr>
		<tr>
			<td>U-bottom 96-well plate</td>
			<td>Corning</td>
			<td>CLS3897</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>BD Falcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes (5mL)</td>
			<td>BD Falcon</td>
			<td>14959-5</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene conical tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene conical tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoir</td>
			<td>USA Scientific</td>
			<td>2321-2230</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NK cell enrichment cocktail</td>
			<td>StemCell Technologies (RosetteSep)</td>
			<td>15065</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based cytotoxicity assay for the assessment of human nk cell activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

Before setting up the assay, it is highly recommended that NK cell content be assessed in the effector population of choice. Figure 1 shows a typical CD56 staining before (light blue) and after (red) NK cell enrichment. NK cells comprise up to 15% of PBMCs and should be at least 80% pure after enrichment.
Flow cytometric analysis in this assay involves detection of two parameters: CFSE, detectable i...

Watch this Scientific Journal Video about A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity at JoVE.com

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

The overall goal of this procedure is to assess the cytotoxic activity of human NK cells. This method can be used to monitor patients with either functional or classic NK cell deficiency. The main advantages of this flow cytometry based technique are that it's sensitive, reproducible, and faster than standard NK cell function assessment methods such as the radioactive chromium release assay.This method can be used to test novel NK cell targeted therapies as well as previously unachievable in depth multi-parameter flow cytometric analysis of NK cell biology. Begin by using density gradient separation to isolate the peripheral blood mononuclear cells, or PBMC's from whole blood samples according to standard protocols. Using PBMC's instead of purified NK cells is greatly beneficial when dealing with clinical specimens.Dilute the isolated cells to a five times 10 to the sixth cells per milliliter concentration in complete medium and immediately collect K562 target cells by centrifugation. Re-suspend five times 10 to the fifth 562 cells in 500 microliters of fresh medium supplemented with one percent FBS. Next label the target cells with 500 microliters of CFSE working solution and gently mix the cell solution by pipetting three to five times.After 10 minutes at 37 degrees Celsius protected from light, quench the labeling reaction with 10 milliliters of complete medium for 10 minutes in the dark at room temperature. At the end of the quenching incubation collect the cells by centrifugation and re-suspend the labeled target cell palette in one milliliter of complete medium for counting. Then dilute the cells to one times 10 to the fifth cells per milliliter of fresh complete medium concentration and place the target cells in a humidified 37 degree Celsius and five percent carbon dioxide incubator.For a successful assay, reduce the target cell stress to a minimum and test the effector cell as soon as possible after collection. Within 30 minutes of the target cell labeling, label a 96 well U bottom tissue culture treated plate according to the table. Next add 100 microliters of complete medium to the 25 to one, 12.5 to one, and 6.25 to one effector to target cell ratio wells, the target cells only wells, and the effector cells only wells.Add 100 microliters of effector cells to the cytotoxcity control wells and the 50 to one, and 25 to one effector cell to target cell ratio wells. Then starting with the 25 to one effector cell to target cell ratio well, transfer 100 microliters of cells to the well with the next lowest cell ratio concentration for the next two wells, skipping the target cell only wells, mixing well between each cell transfer and adding the last 100 microliters of cells the the effector cells only wells. Add 100 microliters of 2X 0.2 percent Tween working solution to the Tween only K562 death positive control wells.Add 30 microliters of vial two working solution to the NK cell cytotoxicity positive control wells. Then add one times 10 to the fifth labeled K562 target cells per milliliter in 100 microliters of complete medium to all of the wells except the effector cells only wells. When all the cells have been plated mix all the wells with the multi channel pipette.Settle the cells with a brief centrifugation and place the plate in the cell culture incubator for four hours. At the end of the incubation place the plate on ice or on a cold platform, and add 50 microliters of freshly prepared dead cell stain to each well. Then gently mix all of the wells and analyze the cells by flow cytometry.Flow cytometric analysis of CSFE and dead cell stain after four hours of cold culture as just demonstrated reveals that the cells in the target cells only wells experience 15%cell death or less. No CSFE signal is detected in the effector cells only wells if less than five percent cell death observed in these wells. While the cell death in the Tween mediated target cell killing wells is typically over 85%In patients with a defect in NK cell function a reduced killing activity is observed at most or all of the tested ratios compared to healthy individuals demonstrating that the NK cells from these patients exhibit a differential reduction in cytotoxicity that correlates with a decreasing effector target cell ratio.Once mastered this technique can be completed in six and a half hours, including the two hours of preparation and plating, four hours of incubation, and 30 minutes of acquisition. While attempting this procedure it's important to remember to prepare the target and effector cells in parallel to minimize the lag time between the cell preparation and the start of the co-incubation. After watching this video you should have a good understanding of how to measure the cytotoxic activity of human NK cells against fluorescently labeled target cells by flow cytometry.

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Research

JoVE Journal

Immunology and Infection

23.3K vistas.  University of California, Los Angeles. The overall goal of this procedure is to assess the cytotoxic activity of human NK cells. This method can be used to monitor patients with either functional or classic NK cell deficiency. The main advantages of this flow cytometry based technique are that it's sensitive, reproducible, and faster than standard NK cell function assessment methods such as the radioactive chromium release assay.This method can be used to test novel NK cell targeted therapies as well as previously unachieva...