The authors thank Dr. Michael N. Sack (National Heart, Lung, and Blood Institute) for support and discussion. This study was supported by the Intramural Research Programs of the National Institutes of Health, National Cancer Institute and National Heart, Lung, and Blood Institute.&#160;JT is supported by the Ramon y Cajal program (grant RYC2018-026050-I) of MICINN (Spain).

All the experiments were performed in accordance with the Declaration of Helsinki&#8217;s ethical principles of medical research. Peripheral blood samples from donors were obtained from the NIH Department of Transfusion Medicine under the 99-CC-0168 IRB approved protocol, with patient written informed consent.
1. Reagent preparation
<ol>
	<li>Reagents for the isolation of NK cells 
	NOTES: Prepare these reagents in a cell culture hood.
	<ol>
		<li>Prepare NK separation ...

<ol>
	<li>Long, E. O., Kim, H. S., Liu, D., Peterson, M. E., Rajagopalan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+natural+killer+cell+responses:+integration+of+signals+for+activation+and+inhibition.">Controlling natural killer cell responses: integration of signals for activation and inhibition.</a> Annual Review of Immunology. 31, 227-258 (2013).</li><li>Caligiuri, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+natural+killer+cells.">Human natural killer cells.</a> Blood. 112 (3), 461-469 (2008).</li><li>Anton, O. M., Vielkind, S., Peterson, M. E., Tagaya, Y., Long, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+Cell+Proliferation+Induced+by+IL-15+Transpresentation+Is+Negatively+Regulated+by+Inhibitory+Receptors.">NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.</a> Journal of Immunology. 195 (10), 4810-4821 (2015).</li><li>Henney, C. S., Kuribayashi, K., Kern, D. E., Gillis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-2+augments+natural+killer+cell+activity.">Interleukin-2 augments natural killer cell activity.</a> Nature. 291 (5813), 335-338 (1981).</li><li>Anton, O. M., 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=Trans-endocytosis+of+intact+IL-15Ralpha-IL-15+complex+from+presenting+cells+into+NK+cells+favors+signaling+for+proliferation.">Trans-endocytosis of intact IL-15Ralpha-IL-15 complex from presenting cells into NK cells favors signaling for proliferation.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (1), 522-531 (2020).</li><li>Marcais, 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=The+metabolic+checkpoint+kinase+mTOR+is+essential+for+IL-15+signaling+during+the+development+and+activation+of+NK+cells.">The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.</a> Nature Immunology. 15 (8), 749-757 (2014).</li><li>Mookerjee, S. A., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+Maximum+Glycolytic+Capacity+Using+Extracellular+Flux+Measurements.">Determining Maximum Glycolytic Capacity Using Extracellular Flux Measurements.</a> PloS One. 11 (3), 0152016 (2016).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>Cheng, 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=Mitochondrial+Proton+Leak+Plays+a+Critical+Role+in+Pathogenesis+of+Cardiovascular+Diseases.">Mitochondrial Proton Leak Plays a Critical Role in Pathogenesis of Cardiovascular Diseases.</a> Advances in Experimental Medicine and Biology. 982, 359-370 (2017).</li><li>Terren, I., Orrantia, A., Vitalle, J., Zenarruzabeitia, O., Borrego, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+Cell+Metabolism+and+Tumor+Microenvironment.">NK Cell Metabolism and Tumor Microenvironment.</a> Frontiers in Immunology. 10, 2278 (2019).</li><li>Felices, M., 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=Continuous+treatment+with+IL-15+exhausts+human+NK+cells+via+a+metabolic+defect.">Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect.</a> Journal of Clinical Investigation Insight. 3 (3), 96219 (2018).</li><li>Miller, J. 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=A+First-in-Human+Phase+I+Study+of+Subcutaneous+Outpatient+Recombinant+Human+IL15+(rhIL15)+in+Adults+with+Advanced+Solid+Tumors.">A First-in-Human Phase I Study of Subcutaneous Outpatient Recombinant Human IL15 (rhIL15) in Adults with Advanced Solid Tumors.</a> Clinical Cancer Research. 24 (7), 1525-1535 (2018).</li><li>Conlon, K. 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=IL15+by+Continuous+Intravenous+Infusion+to+Adult+Patients+with+Solid+Tumors+in+a+Phase+I+Trial+Induced+Dramatic+NK-Cell+Subset+Expansion.">IL15 by Continuous Intravenous Infusion to Adult Patients with Solid Tumors in a Phase I Trial Induced Dramatic NK-Cell Subset Expansion.</a> Clinical Cancer Research. 25 (16), 4945-4954 (2019).</li><li>Dubois, 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=IL15+Infusion+of+Cancer+Patients+Expands+the+Subpopulation+of+Cytotoxic+CD56(bright)+NK+Cells+and+Increases+NK-Cell+Cytokine+Release+Capabilities.">IL15 Infusion of Cancer Patients Expands the Subpopulation of Cytotoxic CD56(bright) NK Cells and Increases NK-Cell Cytokine Release Capabilities.</a> Cancer Immunology Research. 5 (10), 929-938 (2017).</li><li>Traba, 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=Fasting+and+refeeding+differentially+regulate+NLRP3+inflammasome+activation+in+human+subjects.">Fasting and refeeding differentially regulate NLRP3 inflammasome activation in human subjects.</a> Journal of Clinical Investigation. 125 (12), 4592-4600 (2015).</li><li>Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K., Akkaya, 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+Optimized+Protocol+to+Analyze+Glycolysis+and+Mitochondrial+Respiration+in+Lymphocytes.">An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes.</a> Journal of Visualized Experiments. (117), e54918 (2016).</li><li>Horan, M. P., Pichaud, N., Ballard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+quantifying+mitochondrial+dysfunction+in+complex+diseases+of+aging.">Review: quantifying mitochondrial dysfunction in complex diseases of aging.</a> Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 67 (10), 1022-1035 (2012).</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> Journal of Visualized Experiments. (116), e54615 (2016).</li><li>Theorell, J., Bryceson, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Intracellular+Ca(2+)+Mobilization+in+Human+NK+Cell+Subsets+by+Flow+Cytometry.">Analysis of Intracellular Ca(2+) Mobilization in Human NK Cell Subsets by Flow Cytometry.</a> Methods in Molecular Biology. 1441, 117-130 (2016).</li><li>Mookerjee, S. A., Gerencser, A. A., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+intracellular+rates+of+glycolytic+and+oxidative+ATP+production+and+consumption+using+extracellular+flux+measurements.">Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements.</a> Journal of Biological Chemistry. 292 (17), 7189-7207 (2017).</li><li>O'Brien, K. L., Finlay, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunometabolism+and+natural+killer+cell+responses.">Immunometabolism and natural killer cell responses.</a> Nature Reviews: Immunology. 19 (5), 282-290 (2019).</li><li>Ahn, B. H., 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=A+role+for+the+mitochondrial+deacetylase+Sirt3+in+regulating+energy+homeostasis.">A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (38), 14447-14452 (2008).</li><li>Stram, A. R., Payne, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+in+mitochondria:+protein+signaling+in+the+powerhouse.">Post-translational modifications in mitochondria: protein signaling in the powerhouse.</a> Cellular and Molecular Life Sciences. 73 (21), 4063-4073 (2016).</li><li>Armstrong, J. 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=Oxidative+stress+alters+mitochondrial+bioenergetics+and+modifies+pancreatic+cell+death+independently+of+cyclophilin+D,+resulting+in+an+apoptosis-to-necrosis+shift.">Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift.</a> Journal of Biological Chemistry. 293 (21), 8032-8047 (2018).</li><li>Mookerjee, S. A., Goncalves, R. L. S., Gerencser, A. A., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contributions+of+respiration+and+glycolysis+to+extracellular+acid+production.">The contributions of respiration and glycolysis to extracellular acid production.</a> Biochimica et Biophysica Acta. 1847 (2), 171-181 (2015).</li><li>Mookerjee, S. A., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+and+Analysis+of+Extracellular+Acid+Production+to+Determine+Glycolytic+Rate.">Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate.</a> Journal of Visualized Experiments. (106), e53464 (2015).</li><li>Lichtshtein, D., Kaback, H. R., Blume, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+lipophilic+cation+for+determination+of+membrane+potential+in+neuroblastoma-glioma+hybrid+cell+suspensions.">Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (2), 650-654 (1979).</li><li>Michelet, X., 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=Metabolic+reprogramming+of+natural+killer+cells+in+obesity+limits+antitumor+responses.">Metabolic reprogramming of natural killer cells in obesity limits antitumor responses.</a> Nature Immunology. 19 (12), 1330-1340 (2018).</li></ol>

In this paper, we have established a protocol for efficiently isolating and culturing pure and viable primary human NK cells from peripheral blood. We have also optimized the conditions for the measurement of the metabolic activity of these NK cells assessed by oxygen consumption rate and extracellular acidification rate by using an extracellular flux analyzer. Compared to other respirometric methods, the extracellular flux analyzer is fast, requires small numbers of cells, and allows high throughput screenings. However,...

Natural Killer (NK) cells are innate lymphocytes that mediate anti-tumor and anti-viral responses. NK cells comprise 5-15% of all lymphocytes in human peripheral blood, and can be also found in spleen, liver, bone marrow and lymph nodes. NK cells do not express polymorphic clonotypic receptors, such as T-cell receptors (TCR) or B-cell receptors (BCR). In contrast, the activation of their cytolytic functions is prompted by the engagement of receptors that recognize invariable ligands on the surface of a target cell1,2.
Resting human NK cells isolated from peripheral blood can survive for several days in culture medium supplemented with human serum. Activation of NK cells by cytokines such as IL-15 or IL-2 drives the cells to proliferation and to an increase of their killing ability, amongst other effects3,4,5. Several studies have shown a direct correlation between NK cell activation and changes in their metabolic activity6. These metabolic changes are destined to meet the particular requirements of the cells in terms of energy and biosynthesis.
Aerobic cells and organisms obtain energy through a series of chemical reactions that involve the catabolism and oxidation of carbohydrates, fat and proteins. Through a combination of glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, eukaryotic cells meet the majority of their ATP demand and obtain intermediates required as building blocks for macromolecules essential for cell growth and proliferation. The process of glycolysis (Figure 1A) starts with the entry of glucose in the cell. In the cytosol, glucose is phosphorylated and transformed into pyruvate (with a net production of 2 molecules of ATP per glucose molecule), which can be reduced to lactate or transported into the mitochondria to be transformed into Acetyl-CoA and enter the TCA cycle. The TCA cycle continues cycling fueled with more molecules of Acetyl-CoA and produces CO2 (that eventually will diffuse outside the cell and, by reacting with H2O in the medium, will generate carbonic acid that will lead to the acidification of the medium) and NADH, the molecule in charge of donating electrons to the electron transport chain (ETC). The electrons travel through different protein complexes and are finally accepted by oxygen. These complexes (I, III and IV) also pump H+ from the mitochondrial matrix into the intermembrane space. As a consequence of the electrochemical gradient generated, the H+ will enter again to the matrix through the complex V (ATP-synthase), investing the potential energy accumulated into the generation of ATP.
Both glycolysis and mitochondrial respiration can be blocked at different points by using inhibitors. The knowledge and usage of these inhibitors was the basis for the development of the extracellular flux assay. By measuring two simple parameters in real time such as pH and oxygen, the extracellular flux analyzer infers the rate of glycolysis and mitochondrial respiration in a 96-well plate. The glycolysis stress test is performed in a basal medium without glucose (Figure 1B)7. The first measurements of the extracellular acidification rate (ECAR) are indicative of glycolysis-independent acidification. It is referred to as non-glycolytic acidification and correlates with CO2 produced by the TCA that, as explained before, combines with H2O in the medium to generate H+ (TCA-linked ECAR). The first injection is glucose to induce glucose utilization and boost glycolysis. The second injection combines both rotenone, a Complex I inhibitor, and antimycin A, a Complex III inhibitor together, to block the ETC. Cells respond to this dramatic decrease in mitochondrial ATP production by activating glycolysis to maintain cellular ATP levels, and this represents the amount of glycolysis that is not used by the cell in the basal state but could be potentially recruited in response to increases in ATP demand (compensatory glycolysis). The third injection is the glucose analog 2-Deoxyglucose (DG), which competes with glucose as a substrate for the enzyme hexokinase. The product of this phosphorylation, 2-deoxyglucose-6-phosphate cannot be transformed into pyruvate, and therefore glycolysis is blocked, which lowers the ECAR to its minimum. The ECAR measured at this point includes other sources of extracellular acidification that are not attributed to glycolysis or respiratory activity as well as any residual glycolysis not fully inhibited by 2-DG (post 2-DG-acidification).
The mitochondrial stress test is performed in a medium with glucose (Figure 1C)8. The first measurements of the oxygen consumption rate (OCR) correspond to the base line of mitochondrial respiration (basal respiration). The first injection is oligomycin, which inhibits the return of protons through the ATP synthase (complex V), blocking ATP synthesis and thus rapidly hyperpolarize the mitochondrial membrane, which prevents further proton pumping through respiratory complexes, and leads to a decrease in OCR. The comparison between the baseline respiration and the value given by addition of oligomycin represents the ATP-linked respiration. The remaining oligomycin-insensitive rate of oxygen consumption is called proton leak, which represents the flow of protons through the lipid bilayer or proteins in the inner mitochondrial membrane such as the adenine nucleotide translocase9. The second injection is the uncoupler 2,4-dinitrophenol (DNP), an ionophore that induces a massive entry of H+ into the mitochondrial matrix, which leads to depolarization of the mitochondrial membrane and disruption of mitochondrial ATP synthesis. Cells respond to the dissipation of the proton-motive force by increasing the rate of electron transport and oxygen consumption to maximum levels in a futile attempt to recover membrane potential (maximal respiratory capacity). The difference between the maximal respiratory capacity and the basal respiration is the spare respiratory capacity of the cell, which represents the amount of respiration that is not used by the cell to generate ATP in the basal state but could be potentially recruited in response to increases in ATP demand or under conditions of stress8. The third injection is a combination of rotenone and antimycin A. This injection completely stops the ETC and OCR decreases to its lowest level, with the remaining oxygen consumption being non-mitochondrial (caused by NADPH-oxidases, etc.).
Changes in metabolic pathways could somehow predict the functioning of NK cells, as it has been suggested that continuous activation of NK cells with cytokines in vitro could lead to NK cell exhaustion by the study of different metabolic pathways10,11. The correlation between NK cell metabolic status and function is very important from the point of view of cancer immunotherapy. In this field, activation of NK cells with infusion of IL-15, alone or in combination with monoclonal therapeutic antibodies have been tested in order to improve tumor cell killing12,13,14. The knowledge of the metabolic status of the NK cells in response to these treatment strategies would provide a valuable predictor of NK cell activation status and killing function.
The study of metabolic pathways in other myeloid and lymphoid cells such as monocytes, T and B cells has been described15 and optimized methods have been published16. In this protocol we provide a method that combines both an NK isolation protocol that yields high numbers of pure and viable NK cells and an optimized protocol to measure metabolic activity using an extracellular flux analyzer. Here we show that this is a valid method for the study of metabolic changes in resting and IL-15 activated human NK cells. For the extracellular flux assay, parameters such as cell number and drug concentrations have been tested and optimized. Compared with other respirometric methods, the extracellular flux analyzer is fully automated and able to test in real time, with very low quantities of cells, up to 92 samples simultaneously, and thus allows high throughput screenings (with multiple samples and replicates) in a relatively quick manner17.
This method can be used by researchers interested in assessing NK cell function by studying NK cell metabolism. It could be applied as well to cells activated by other cytokines, antibodies or soluble stimuli.

<table>
	<tbody>
		<tr>
			<td>2-Deoxy-D-glucose (2-DG)</td>
			<td>MilliporeSigma</td>
			<td>D8375-5G</td>
			<td>Glycolyisis stress test injector compound</td>
		</tr>
		<tr>
			<td>2,4-Dinotrophenol (2,4-DNP)</td>
			<td>MilliporeSigma</td>
			<td>D198501</td>
			<td>ETC uncoupler / mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>96 Well Cell Culture Plate/ Round bottom with Lid</td>
			<td>Costar</td>
			<td>3799</td>
			<td>NK cell culture</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>MilliporeSigma</td>
			<td>A8674</td>
			<td>Complex III inhibitor / glycolysis and mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>BD FACSDIVA Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow data acquisition</td>
		</tr>
		<tr>
			<td>BD LSR Fortessa</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow data acquisition</td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Cell adhesive</td>
		</tr>
		<tr>
			<td>CyQUANT cell proliferation assay</td>
			<td>ThermoFisher Scientific</td>
			<td>C7026</td>
			<td>Cell proliferation Assay for DNA quantification. Contains cell-lysis buffer and CyQUANT GR dye</td>
		</tr>
		<tr>
			<td>EasySep Human CD3 Positive Selection Kit II</td>
			<td>Stemcell technologies</td>
			<td>17851</td>
			<td>NK cell isolation from PBMCs</td>
		</tr>
		<tr>
			<td>EasySep Human NK cell Enrichment Kit</td>
			<td>Stemcell technologies</td>
			<td>19055</td>
			<td>NK cell isolation from PBMCs</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stemcell technologies</td>
			<td>18001</td>
			<td>NK cell isolation from PBMCs</td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8</td>
			<td>Quality Biological</td>
			<td>10128-446</td>
			<td>NK sell separation buffer</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Falcon-Fisher Scientific</td>
			<td>352235</td>
			<td>Flow cytometry experiment</td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical tubes</td>
			<td>Falcon-Fisher Scientific</td>
			<td>14-432-22</td>
			<td>NK cell separation</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>NK cell separation buffer</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow data analysis</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>MilliporeSigma</td>
			<td>G8270</td>
			<td>Component of mitochondrial stress test medium. Glycolysis stress test injector compound</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>78429</td>
			<td>Protease inhibitor 100X. Use in RIPA lysis buffer</td>
		</tr>
		<tr>
			<td>Human IL-15</td>
			<td>Peprotech</td>
			<td>200-15-50ug</td>
			<td>NK cell stimulation</td>
		</tr>
		<tr>
			<td>Human serum (HS)</td>
			<td>Valley Biomedical</td>
			<td>9C0539</td>
			<td>NK cell culture medium supplement</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td>NK cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>25030-081</td>
			<td>Component of stress test media</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>L34965</td>
			<td>Viability dye for flow cytometry staining</td>
		</tr>
		<tr>
			<td>LSM</td>
			<td>mpbio</td>
			<td>50494X</td>
			<td>PBMCs separation from human blood</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD3 BV711</td>
			<td>BD Biosciences</td>
			<td>563725</td>
			<td>T cell flow cytometry staining</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD56 PE</td>
			<td>BD Pharmingen</td>
			<td>555516</td>
			<td>NK flow cytometry staining</td>
		</tr>
		<tr>
			<td>Mouse anti-human NKp46 PE</td>
			<td>BD Pharmingen</td>
			<td>557991</td>
			<td>NK flow cytometry staining</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>MilliporeSigma</td>
			<td>75351</td>
			<td>Complex V inhibitor / mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>NK cell separation buffer</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td>For determination of protein concentration</td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Boston BioProducts</td>
			<td>BP-115</td>
			<td>Cell lysis</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>MilliporeSigma</td>
			<td>R8875</td>
			<td>Complex II inhibitor / glycolysis and mitochondrial stress test injector compound</td>
		</tr>
		<tr>
			<td>Seahorse Wave Controller Software</td>
			<td>Agilent</td>
			<td></td>
			<td>Controller for the Seahorse XFe96 Analyzer</td>
		</tr>
		<tr>
			<td>Seahorse Wave Desktop Software</td>
			<td>Agilent</td>
			<td></td>
			<td>For data analysis</td>
		</tr>
		<tr>
			<td>Seahorse XF Base Medium</td>
			<td>Agilent</td>
			<td>102353-100</td>
			<td>Extracellular Flux assay base medium</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Extracellular Flux Analyzer</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent</td>
			<td>102416-100</td>
			<td>Includes 20 XF96 cell culture plates, 18 XFe96 sensor cartridges, loading guides for transferring compounds to the assay cartridge, and 1 bottle of calibrant solution (500 ml).</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>MilliporeSigma</td>
			<td>S5761</td>
			<td>To prepare the Cell-Tak solution</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11360-070</td>
			<td>Component of mitochondrial stress test medium</td>
		</tr>
	</tbody>
</table>

analysis of human natural killer cell metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

Isolation of NK cells from peripheral blood provides a pure and viable population
The extracellular flux assay is based on the measurement of H+ and O2 concentration in the well and will not distinguish among different populations of cells or their viability. For this reason, obtaining a highly pure and viable population of the cell of interest was the key step to succeed in these experiments.
The isolation of NK cells from per...

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Analysis of Human Natural Killer Cell Metabolism

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

This method allows for the determination of natural killer cell metabolism. A key parameter in the activation by measuring oxygen consumption and pH changes in the extracellular medium. The advantage of the extracellular flux analyzer is that it is fully automated and able to test up to 92 samples in real time with low quantities of cells.Thus allowing high throughput screens. A valid method to determine glycolysis and respiration in inky cells is very important in the clinic. Since there are many diseases including obesity and cancer where inky cell metabolism is impaired.Begin by pipetting 20 milliliters of lymphocyte separation medium into a 50 milliliter conical tube. While keeping the tube at 30 degree angle, gently pipette 20 milliliters of blood over the medium. Creating a visible and well-defined interface between the two fluids.Centrifuge the tubes for 25 minutes at 1000 times G.Then carefully take the tubes out of the centrifuge and place them in a rack. Check for the presence of a conspicuous layer of cells at the interface between LSM and plasma. Gently aspirate the mononuclear cell layer with a 10 milliliter plastic pipette and place it in a new 50 milliliter conical tube.Wash the mononuclear cells twice with 45 milliliters of PBS. And centrifuge them at 800 times G for five minutes. To isolate the natural killer cells or engaves, count the PVM seas and resuspend them in NK separation buffer at one times 10 to the eighth cells per milliliter.Then transfer 10 milliliters of the cell suspension into a new 50 milliliter tube. Add 500 microliters of NK cell isolation antibody mix. And 10 microliters of anti CD three positive isolation antibody mix to the PBMCs.And incubate them at room temperature for 10 minutes. Vortex magnetic beads and add one milliliter to the PBMCs and antibodies. Then incubate the MIGS for 10 minutes at room temperature with occasional stirring.Add 35 milliliters of NK isolation buffer and place the tube on the magnet for 15 minutes. Carefully collect the supernatant with a 50 milliliter plastic pipette without touching the sides or the bottom of the tube. Count the cells and centrifuge them at 800 times G for five minutes.To stimulate the NK cells with soluble aisle 15, resuspend 750, 000 cells in 100 microliters of IMDM containing 10%HS in a well of a 96 well plate. Dilute human aisle 15 to one microgram per milliliter in the IMDM and 10%HS. And add 100 microliters of the diluted human aisle 15 to the cells.Repair a control sample with unstimulated cells and place both samples in the incubator at 37 degrees Celsius. Stimulate the cells for 48 hours then perform the extracellular flux assay. The day before the experiment, turn on the analyzer and let it warm up to 37 degrees Celsius.Open the censor cartridge package and separate the cartridge from the utility plate. Then add 200 microliters of the calibrant solution to each well of the utility plate. And put the central cartridge back in making sure that the sensors are completely submerged.For optimum results incubate the cartridge overnight at 37 degrees Celsius in a carbon dioxide free incubator. Which properly humidified. To prepare an adhesive coated blade, pipette 25 microliters of the cell adhesive solution to each well of the plate.And incubated at room temperature for 20 minutes. Then remove the solution and wash the plate twice with 200 microliters of sterile water per well. Keep the plate open for 15 minutes inside the cell culture hood to allow the wells to dry.Centrifuge the previously isolated NK cells add 200 times G for five minutes. Then remove supernatants and wash the cells in warmed mitochondrial stress test medium or glycolysis stress test medium. Pellet the cells again and resuspend them to the preferred cell concentration in the same medium.Lay 180 microliters of cell suspension per well into the assay plate. Use wells A one, A 12 H one and H 12 as control wells for background correction. Adding 180 microliters of the assay medium to these Wells Incubate the plate for 30 minutes at 37 degrees Celsius in a carbon dioxide free incubator.Centrifuge the plate at 200 times G for five minutes. Then observe the cells under the microscope to check that they form a mano layer at the bottom of the well. Incubate the cells for another 25 minutes.Warm working solutions to 37 degrees Celsius. And readjust their pH to 7.4 if needed. Load the previously prepared compounds for a mitochondrial stress test or for a glycolysis stress test into boards A, B and C of the hydrated censor cartridge Put the loaded censor cartridge back in the incubator while setting up the program.To set up the extracellular flux assay protocol, open the software and use Group definitions to define the pretreatment conditions. Use the Plate map tab to indicate the groups of wells that have similar conditions Also indicate the background correction and empty wells. Then set the program in the protocols tab.Start the program and place the sensor cartridge and utility plate onto the tray. After the calibration step, replace the calibrant plate for the assay plate with the attached cells. Once the run is completed, retrieve the data and analyze it.In order to assess the purity and viability of the NK cells. Small Alec Watts from PVM seas and the isolated NK cell population were stained and analyzed by flow cytometry. The purity of the NK cell population was established by double staying against CD three and CD 56 or NKP 46.Lots of mitochondrial oxygen consumption rate for the human NK cells are shown here. As expected I cell numbers displayed higher OCR values. Cell numbers also correlated linearly with the amount of DNA or protein in the well.On the other hand higher cell numbers were not optimal. Because upon addition of DNP, the oxygen concentration in the well was totally depleted in each cycle. Which prevented the accurate calculation of the OCR.In human NK cells, 100 micromolar DNP was found to be the optimal dose. A typical mitochondrial stress test experiment with 750, 000 NK cells per well is shown here. In this test Oligomycin leads to a dramatic decrease in oxygen consumption.And an increase in ECAR. Which represents a switch to glycolysis and an effort to maintain cellular ATP levels. Activation of the NK cells by aisle 15 caused an increase in both mitochondrial oxygen consumption and extracellular acidification.Basal Maximal and ATP linked respiration increased but not proton leak or non mitochondrial respiration. Furthermore, the OCR/ECA rate decreased Indicating a shift to a glycolytic metabolism after IL 15 stimulation. When performing this protocol, it is very important to obtain a pure and healthy cell population to determine the optimal cell number and to titrate the concentration of uncablers.

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JoVE Journal

Immunology and Infection

6.7K visualizações.  National Institutes of Health. Este método permite a determinação do metabolismo celular assassino natural. Um parâmetro-chave na ativação medindo o consumo de oxigênio e as alterações de pH no meio extracelular. A vantagem do analisador de fluxo extracelular é que ele é totalmente automatizado e capaz de testar até 92 amostras em tempo real com baixas quantidades de células.Permitindo assim telas de alto rendimento. Um método válido para determinar a glicólise e a respiração em células inky é mu...