Name: a flow cytometry-based cytotoxicity assay for the assessment of human nk cell activity
Uploaded: 2017-08-09T14:00:00
Description: Watch this Scientific Journal Video about A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity at JoVE.com

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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W. <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+cell+cytotoxicity:+how+do+they+pull+the+trigger?.">Natural killer cell cytotoxicity: how do they pull the trigger?.</a> Immunology. 128 (1), 7-15 (2009).</li><li>Warren, H. S., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cells+and+apoptosis.">NK cells and apoptosis.</a> Immunol Cell Biol. 77 (1), 64-75 (1999).</li><li>Tognarelli, S., Jacobs, B., Staiger, N., Ullrich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry-based+Assay+for+the+Monitoring+of+NK+Cell+Functions.">Flow Cytometry-based Assay for the Monitoring of NK Cell Functions.</a> J Vis Exp. (116), (2016).</li><li>Somanchi, S. S., McCulley, K. J., Somanchi, A., Chan, L. L., Lee, D. A. <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+Method+for+Assessment+of+Natural+Killer+Cell+Cytotoxicity+Using+Image+Cytometry.">A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.</a> PLoS One. 10 (10), e0141074 (2015).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J Immunol Methods. 294 (1-2), 15-22 (2004).</li><li>Atkinson, E. A., Gerrard, J. M., Hildes, G. E., Greenberg, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+mechanism+of+natural+killer+(NK)+degranulation+and+cytotoxicity.">Studies of the mechanism of natural killer (NK) degranulation and cytotoxicity.</a> J Leukoc Biol. 47 (1), 39-48 (1990).</li><li>Kim, G. G., Donnenberg, V. S., Donnenberg, A. D., Gooding, W., Whiteside, T. 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+multiparametric+flow+cytometry-based+cytotoxicity+assay+simultaneously+immunophenotypes+effector+cells:+comparisons+to+a+4+h+51Cr-release+assay.">A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.</a> J Immunol Methods. 325 (1-2), 51-66 (2007).</li><li>Kane, K. L., Ashton, F. A., Schmitz, J. L., Folds, J. 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 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...

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, ...

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