Name: flow cytometry-based assay for the monitoring of nk cell functions
Uploaded: 2016-10-30T15:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Authors were supported by the German Cancer Aid (Max Eder Nachwuchsgruppe, Deutsche Krebshilfe; EU), the LOEWE Center for Cell and Gene Therapy Frankfurt (EU, ST) funded by the Hessian Ministry of Higher Education, Research and the Arts, Germany (III L 4- 518/17.004) and by the "Alfred- und Angelika Gutermuth-Stiftung", Frankfurt, Germany (EU). BJ was funded by a Mildred Scheel postdoctoral scholarship from the Dr. Mildred Scheel Foundation for Cancer Research. ST was founded by a GO-IN postdoctoral fellowship (PCOFUND-GA-2011-291776). The authors thank Becton Dickinson (BD) for providing the FACSCanto II and Canto10c Flow Cytometry Analyzers used in this study.

This study was carried out in accordance with the recommendations of the local ethics committee of the University of Frankfurt.
1. Culturing of K562 Cells
<ol>
	<li>Culture K562 cells in R10 media (RPMI1640 with glutamine medium, 1% penicillin/streptomycin, 10% fetal calf serum) at a density of 0.5-1 x 106 cells per ml in a cell culture flask at 37 &#176;C and 5% CO2.</li>
	<li>Harvest K562 Cells 24 hr Before the Start of a New Experiment.
	<ol>
		<li>Remove the cell culture flask containin...

<ol>
	<li>Watzl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+trigger+a+killer:+modulation+of+natural+killer+cell+reactivity+on+many+levels.">How to trigger a killer: modulation of natural killer cell reactivity on many levels.</a> Adv Immunol. 124, 137-170 (2014).</li><li>Khaznadar, Z., 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=Defective+NK+Cells+in+Acute+Myeloid+Leukemia+Patients+at+Diagnosis+Are+Associated+with+Blast+Transcriptional+Signatures+of+Immune+Evasion.">Defective NK Cells in Acute Myeloid Leukemia Patients at Diagnosis Are Associated with Blast Transcriptional Signatures of Immune Evasion.</a> J Immunol. 195 (6), 2580-2590 (2015).</li><li>Pical-Izard, C., 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=Reconstitution+of+natural+killer+cells+in+HLA-matched+HSCT+after+reduced-intensity+conditioning:+impact+on+clinical+outcome.">Reconstitution of natural killer cells in HLA-matched HSCT after reduced-intensity conditioning: impact on clinical outcome.</a> Biol Blood Marrow Transplant. 21 (3), 429-439 (2015).</li><li>Ahlenstiel, G., 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=Early+changes+in+natural+killer+cell+function+indicate+virologic+response+to+interferon+therapy+for+hepatitis+C.">Early changes in natural killer cell function indicate virologic response to interferon therapy for hepatitis C.</a> Gastroenterology. 141 (4), 1231-1239 (2011).</li><li>Porrata, L. F., 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=Early+lymphocyte+recovery+predicts+superior+survival+after+autologous+stem+cell+transplantation+in+non-Hodgkin+lymphoma:+a+prospective+study.">Early lymphocyte recovery predicts superior survival after autologous stem cell transplantation in non-Hodgkin lymphoma: a prospective study.</a> Biol Blood Marrow Transplant. 14 (7), 807-816 (2008).</li><li>Silva, I. 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=Reversal+of+NK-cell+exhaustion+in+advanced+melanoma+by+Tim-3+blockade.">Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade.</a> Cancer Immunol Res. 2 (5), 410-422 (2014).</li><li>Kiessling, R., Klein, E., Wigzell, H. <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+the+mouse.+I.+Cytotoxic+cells+with+specificity+for+mouse+Moloney+leukemia+cells.+Specificity+and+distribution+according+to+genotype.">"Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype.</a> Eur J Immunol. 5 (2), 112-117 (1975).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. 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Immunol. 6, 583 (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>Theorell, J., 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=Sensitive+and+viable+quantification+of+inside-out+signals+for+LFA-1+activation+in+human+cytotoxic+lymphocytes+by+flow+cytometry.">Sensitive and viable quantification of inside-out signals for LFA-1 activation in human cytotoxic lymphocytes by flow cytometry.</a> J Immunol Methods. 366 (1-2), 106-118 (2011).</li><li>Brander, C., 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=Inhibition+of+human+NK+cell-mediated+cytotoxicity+by+exposure+to+ammonium+chloride.">Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride.</a> J Immunol Methods. 252 (1-2), 1-14 (2001).</li><li>Beeton, C., Chandy, K. 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R., 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=Serglycin+determines+secretory+granule+repertoire+and+regulates+NK+cell+and+CTL+cytotoxicity.">Serglycin determines secretory granule repertoire and regulates NK cell and CTL cytotoxicity.</a> FEBS J. 283 (5), 947-961 (2016).</li><li>Sch&#246;nberg, K., Hejazi, M., Uhrberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+clonal+analysis+of+NK+cell+effector+functions+by+multi-parameter+flow+cytometry.">Protocol for the clonal analysis of NK cell effector functions by multi-parameter flow cytometry.</a> Methods Mol Biol. 903, 381-392 (2012).</li><li>Bj&#246;rkstr&#246;m, N. 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G., 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=IDEC-C2B8+(Rituximab)+anti-CD20+monoclonal+antibody+therapy+in+patients+with+relapsed+low-grade+non-Hodgkin's+lymphoma.">IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma.</a> Blood. 90 (6), 2188-2195 (1997).</li><li>Salles, G., 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=Phase+1+study+results+of+the+type+II+glycoengineered+humanized+anti-CD20+monoclonal+antibody+obinutuzumab+(GA101)+in+B-cell+lymphoma+patients.">Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients.</a> Blood. 119 (22), 5126-5132 (2012).</li><li>Terszowski, G., Klein, C., Stern, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIR/HLA+interactions+negatively+affect+rituximab-+but+not+GA101+(obinutuzumab)-induced+antibody-dependent+cellular+cytotoxicity.">KIR/HLA interactions negatively affect rituximab- but not GA101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity.</a> J Immunol. 192 (12), 5618-5624 (2014).</li><li>Hsu, A. 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The described method is an easy, fast and reliable approach to study NK cell functions from whole blood samples of healthy donors or patients. This method offers the great advantage to directly purify NK cells from whole blood, avoiding the time-consuming density gradient centrifugation, which is mandatory for many other purification methods15. Moreover, it requires a smaller sample size compared to &#34;classical&#34; NK cell isolation/enrichment methods, which makes it a suitable alternative for samples of p...

As part of the innate immune system natural killer (NK) cells contribute to the first line of defense against virus-infected or malignantly transformed cells. A system of inhibitory and activating receptors enables them to distinguish between healthy and transformed cells without prior antigen priming in contrast to T cells. Upon target cell encounter NK cells release the content of their cytotoxic granules (e.g., perforin, granzymes) into the immune synapse to kill their target. Moreover, NK cells produce and secrete different kinds of cytokines (e.g., interferon- &#947;: IFN-&#947;; tumor necrosis factor-&#945;: TNF-&#945;) and chemokines (e.g., macrophage inflammatory protein-1&#946;: MIP-1&#946;) upon target cell interaction or cytokine stimulation1.
Sufficient NK cell functions such as cytotoxicity, chemokine and cytokine production have an important impact on the fate of diverse diseases. Leukemia patients show increased relapse rates if they exhibit a defective NK cell profile at diagnosis consisting of reduced IFN-&#947; production and reduced expression of activating NK cell receptors2. An early recovery of NK cell numbers and function including cytokine production upon target cell interaction is associated with a reduced relapse and improved survival rate in patients receiving allogeneic stem cell transplantation3. Moreover, upon initiation of interferon therapy in hepatitis C virus-infected patients the degranulation capacity of peripheral NK cells is stronger in early responders than in non-responders4. NK cell numbers (&#62;80/&#181;l) on day 15 after autologous stem cell transplantation (autoSCT) in patients suffering from lymphoma or multiple myeloma are predictive for an improved progression free and overall survival5. In melanoma patients the expression of the T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3), an immune-regulatory protein on NK cells, correlates with disease stage and prognosis6.
Scientists have monitored NK cell functions throughout the last decades. The initial analysis of NK cell cytotoxicity against tumor cells without prior priming was addressed using a 51Cr-release assay7. More recently, scientists developed a non-radioactive method to evaluate the cytotoxicity of expanded NK cells8. Cytokine and chemokine production has been frequently evaluated using enzyme-linked immunosorbent assay (ELISA) techniques9,10. During the last decades these methods have been complemented by flow cytometry-based assays. The use of protein transport inhibitors (e.g., brefeldin A and monensin) and cell permeabilization methods in combination with conventional surface staining protocols have enabled scientists to study chemokine and cytokine production in different specific lymphocyte subsets (e.g., T, B or NK cells)11. Moreover, different flow cytometry-based assays have been developed to monitor T and NK cell cytotoxicity. In 2004 Alter et al. described the surface expression of the lysosome-associated protein CD107a (Lamp1) on NK cells upon target cell encounter as a marker for the degranulation of cytotoxic granules12. Since a wide range of different fluorochromes and multi-channel flow cytometers are available in our days, it has become possible to simultaneously monitor diverse NK cell functions (cytotoxicity, cytokine and chemokine production) in different NK cell subsets. This becomes especially important in situations where sample size is limited, e.g., in biopsies or blood samples of patients suffering from leukopenia.
To test global NK cell functions, the different flow cytometry-based assays can be efficiently combined. Theorell et al. stimulated NK cells from healthy donors with the tumor cell line K562 and analyzed NK cell degranulation, inside-out signal and chemokine production via flow cytometry13. Recently NK cell subgroups, phenotypes and functions in tumor patients during autoSCT were analyzed using flow cytometry-based assays. It was demonstrated that NK cells were able to degranulate and produce cytokines/chemokines upon tumor cell recognition at very early time points after autoSCT11.
Here a protocol is described to evaluate NK cell functions upon interaction with tumor cells including degranulation capacity, chemokine and cytokine production using a flow cytometry-based assay that makes it possible to monitor NK cell functions in different subsets simultaneously.

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			<td height="21" style="height:21px;width:216px;">RPMI 1640 + glutamine</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">6187-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicilin/streptomycin</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Falcon Round Bottom Tube</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">352008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">fetal calf serum</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:119px;">10270-106</td>
			<td style="width:167px;">heat inactivated before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-flask</td>
			<td style="width:135px;">Greiner Bio-One</td>
			<td style="width:119px;">690195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K562 tumor cell line</td>
			<td style="width:135px;">DSMZ GmbH</td>
			<td style="width:119px;">ACC 10</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:216px;">ammonium-chloride-potassium (ACK) lysis buffer</td>
			<td style="width:135px;">made in house</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;">components are listed in the text</td>
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			<td height="42" style="height:42px;width:216px;">Distilled water: Ampuwa Sp&#252;ll&#246;sung 1000ml Plastipur</td>
			<td style="width:135px;">Fresenius Kabi</td>
			<td style="width:119px;">1088813</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Megafuge 40R Centrifuge</td>
			<td>Heraeus</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">EDTA blood collector tubes</td>
			<td style="width:135px;">Sarstedt</td>
			<td style="width:119px;">386453</td>
			<td style="width:167px;">S-Monovette 7,5 ml, K3 EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Hematopoietic media (XVIVO)</td>
			<td>Lonza Group Ltd</td>
			<td style="width:119px;">BE04-743Q</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">human serum</td>
			<td style="width:135px;">DRK Blutspendedienst, Frankfurt/M&#160;</td>
			<td style="width:119px;">/</td>
			<td style="width:167px;">healthy donors with blood type AB; heat inactivated before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Neubauer-improved counting chamber, bright line</td>
			<td style="width:135px;">Marienfeld superior/ LO-Laboroptik Ltd.</td>
			<td style="width:119px;">0640030/ 1110000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trypan blue solution (0,4%)</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3% acetic acid with methylene blue</td>
			<td style="width:135px;">Stemcell Technologies&#160;</td>
			<td style="width:119px;">07060</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 96 Well Clear V-Bottom TC-treated Microplates</td>
			<td style="width:135px;">Corning&#160;</td>
			<td style="width:119px;">3894</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon 96 Well Round Bottom Not Treated Microplates</td>
			<td style="width:135px;">Corning&#160;</td>
			<td style="width:119px;">351177</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">DPBS (Ca2+- and Mg2+-free)</td>
			<td style="width:135px;">Gibco Invitrogen</td>
			<td style="width:119px;">14190-169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">A2153-100G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaN3</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">08591-1ML-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phorbol 12-myristate 13-acetate (PMA)&#160;</td>
			<td style="width:135px;">Merck</td>
			<td style="width:119px;">524400-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ionomycin&#160;</td>
			<td style="width:135px;">PromoKine</td>
			<td style="width:119px;">PK-CA577-1566-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">interleukin 15 (IL-15)</td>
			<td>PeproTech</td>
			<td>200-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proleukin S (IL-2)&#160;</td>
			<td style="width:135px;">Novartis Pharma</td>
			<td style="width:119px;">730523</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Golgi Stop, Protein Transport Inhibitor (containing Monensin)</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">554724</td>
			<td style="width:167px;">This product can be used in combination or instead of Golgi Plug. The best combination for the wished experimental setting has to be tested.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Golgi Plug, Protein Transport Inhibitor (containing Brefeldin A)</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">UN2209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">saponin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">flow cytometer: Canto10C</td>
			<td style="width:135px;">BD Biosciences</td>
			<td style="width:119px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FlowJo</td>
			<td style="width:135px;">TreeStar Inc.</td>
			<td style="width:119px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graph Pad</td>
			<td style="width:135px;">Graph Pad Inc.</td>
			<td style="width:119px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACSxpress Separator</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACSxpress NK isolation kit</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-185</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACSxpress Erythrocyte Depletion Kit, human</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-098-196</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACSmix Tube Rotator&#160;</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td style="width:119px;">130-090-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD3 APC</td>
			<td style="width:135px;">Biolegend</td>
			<td>300412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD3 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD14 PerCP</td>
			<td style="width:135px;">Miltenyi Biotec&#160;</td>
			<td>130-094-969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD14 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD16 PE</td>
			<td style="width:135px;">Biolegend</td>
			<td>302008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD16 PerCP</td>
			<td style="width:135px;">Biolegend</td>
			<td>302029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD19 PE-Cy7</td>
			<td style="width:135px;">Biolegend</td>
			<td>302216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD19 V450</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>560353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD45 BV510</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>563204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD56 FITC</td>
			<td style="width:135px;">Biolegend</td>
			<td>345811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human CD107a PE</td>
			<td style="width:135px;">Biolegend</td>
			<td>328608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human IFN-&#947; AF-647</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>557729</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-human MIP-1&#946; APC-H7</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>561280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:135px;">Biolegend</td>
			<td>422801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zombie Violet Fixable Viability Kit</td>
			<td>Biolegend</td>
			<td>423113</td>
			<td>fixable dead cell marker</td>
		</tr>
	</tbody>
</table>

flow cytometry-based assay for the monitoring of nk cell functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

The gating strategy for analyzing the degranulation, cytokine and chemokine production of the whole NK cell population and three different NK cell subsets are illustrated in Figure 1.
Representative results of one healthy donor are illustrated in Figure 2. NK cells without any stimulus produced neither IFN-&#947; nor MIP-1&#946; and did not express CD107a on their surface (Figure 2A</str...

Watch this Scientific Journal Video about Flow Cytometry-based Assay for the Monitoring of NK Cell Functions at JoVE.com

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

The overall goal of this procedure is to monitor NK cell functions using a flow cytometry-based assay. Natural killer cells are crucial for the outcome of various viral infections and malignant diseases. To better monitor NK cell functions, our lab has further developed a flow cytometry-based assay that can be used in both scientific research and clinical evaluations.We optimized the protocol to permit the analysis of small NK cell sample sizes which is essential for its use in pediatrics and for immunodeficient patients. An additional advantage of this technique is the possibility to measure NK cell functions not only within the whole NK cell population, but also in different NK cell subsets. To isolate the NK cells, begin by mixing the appropriate volume of NK cell isolation cocktail solution with six to 10 milliliters of EDTA peripheral whole blood from a patient by several inversions.Next, further homogenize the sample on a tube rotator for five minutes at room temperature. Then place the tube into a magnetic separator for 15 minutes under sterile conditions, taking care that the tube label faces toward the back of the magnet to facilitate the visibility of the separation line. At the end of the separation, carefully transfer the supernatant into a new 15 milliliter tube and bring the final volume up to 15 milliliters with complete medium.Wash the cells by centrifugation and resuspend the pellet in one milliliter of fresh complete medium. Then count the cells and adjust the volume to a concentration of at least two times 10 to the fourth NK cells per 100 microliters in complete medium. To stimulate the isolated NK cells, first gently mix by pipetting at least five times and aliquot 100 microliters of the cells into the appropriate wells of a 96-well V-bottomed plate.Switch off the light and add anti-CD107a antibody to each well of the NK cells. Then carefully mix an aliquot of K562 tumor cells at a two times 10 to the fourth cells per 100 microliter volume by pipetting at least five times and transfer 100 microliters of the tumor cells to each well planned for the stimulation. Precise pipetting and the preparation of a one-to-one effector target ratio are of key importance for the accurate assessment of NK cell function.Next, add IL-2 and IL-15 to the stimulation wells and mix all of the wells by pipetting at least five times. Incubate the plate protected from light for three hours at 37 degrees Celsius and 5%carbon dioxide. After the first hour with the light switched off, add freshly prepared protein transport inhibitor solution to each well with careful mixing and return the plate to the incubator.At the end of incubation, carefully mix the co-cultures and transfer them into their corresponding flow cytometry tubes. Add one milliliter of wash buffer to each tube for centrifugation and resuspend the pellets in 99 microliters of PBS plus one microliter of fixable dead cell marker per sample. Mix the cells by vortexing, followed by a 15-minute incubation at room temperature in the dark.At the end of the incubation, wash the cells in one milliliter of wash buffer and resuspend the pellets in 84 microliters of wash buffer plus 16 microliters of the appropriate cell surface antibody cocktail. Mix the samples by vortexing and incubate for 20 minutes in the dark at four degrees Celsius. Then wash the cells in one milliliter of wash buffer and resuspend the pellets in 100 microliters of a cold fixation solution.Mix by vortexing, followed by 10 minutes at four degrees Celsius in the dark. Now wash the cells in one milliliter of wash buffer and resuspend the pellets in 90 microliters of permeabilization buffer. Stain with 10 microliters of the appropriate intracellular antibodies and mix by vortexing.After 30 minutes in the dark at four degrees Celsius, wash the cells two times in one milliliter of fresh permeabilization buffer. Then resuspend the samples in 400 microliters of fresh permeabilization buffer. Mix well by vortexing and place the cells on ice until their analysis by flow cytometry.As soon as possible, transfer all tubes into the flow cytometer and start the data acquisition. Here, gating strategies for analyzing the de-granulation and cytokine and chemokine production of the whole NK cell population as well as three different NK cell subsets are illustrated. Unstimulated NK cells from healthy donors produced neither interferon gamma nor MIP-1 beta and do not express CD107a on their surface.In contrast, NK cell stimulated with tumor cells and cytokines produced significant amounts of intracellular interferon gamma and MIP-1 beta with over 20%of the cells demonstrating de-granulation upon tumor cell interaction as indicated by their CD107a cell surface expression. Once mastered, this technique can be completed in eight hours. While attempting this procedure, it is important to be precise in the plating of the NK cells and K562 tumor cells.To maintain the selected effector target ratio, the counting should be as accurate as possible. This flexible procedure can be easily modified. For example, NK cells within the PBMC population can be analyzed or different antibodies can be used to check for other markers of interest.After watching this video, you should have a good understanding of how to purify, stimulate and label NK cells to monitor their function using a flow cytometry-based assay. Don't forget that working with human and target cells is potentially hazardous and that safety precautions such as wearing the appropriate personal protection equipment should always be taken while performing this procedure.

flow-cytometry-based-assay-for-the-monitoring-of-nk-cell-functions

Research

JoVE Journal

Immunology and Infection

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

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.

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, ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...