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

Deze studie werd uitgevoerd in overeenstemming met de aanbevelingen van de lokale ethische commissie van de Universiteit van Frankfurt uitgevoerd. 1. Opkweek K562 cellen <ol><li> Cultuur K562 cellen in R10 medium (RPMI1640 medium met glutamine, 1% penicilline / streptomycine, 10% foetaal kalfsserum) bij een dichtheid van 0,5-1 x 10 6 cellen per ml in een celkweek kolf bij 37 ° C en 5% CO 2. </li><li> Harvest K562 cellen 24 uur voor het begin van een nieuw experiment. <ol><li> Verwijder d...

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	<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> J Vis Exp. (48), (2011).</li><li>Mariani, 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=Chemokine+production+by+natural+killer+cells+from+nonagenarians.">Chemokine production by natural killer cells from nonagenarians.</a> Eur J Immunol. 32 (6), 1524-1529 (2002).</li><li>Reefman, 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=Cytokine+secretion+is+distinct+from+secretion+of+cytotoxic+granules+in+NK+cells.">Cytokine secretion is distinct from secretion of cytotoxic granules in NK cells.</a> J Immunol. 184 (9), 4852-4862 (2010).</li><li>Jacobs, B., 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=NK+Cell+Subgroups,+Phenotype,+and+Functions+After+Autologous+Stem+Cell+Transplantation.">NK Cell Subgroups, Phenotype, and Functions After Autologous Stem Cell Transplantation.</a> Front. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+NK+cells+from+human+blood+with+the+RosetteSep+kit+from+StemCell+technologies.">Enrichment of NK cells from human blood with the RosetteSep kit from StemCell technologies.</a> J Vis Exp. (8), e326 (2007).</li><li>Lamoreaux, L., Roederer, M., Koup, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cytokine+optimization+and+standard+operating+procedure.">Intracellular cytokine optimization and standard operating procedure.</a> Nat. Protoc. 1 (3), 1507-1516 (2006).</li><li>Baginska, 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=Granzyme+B+degradation+by+autophagy+decreases+tumor+cell+susceptibility+to+natural+killer-mediated+lysis+under+hypoxia.">Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.</a> Proc Natl Acad Sci U S A. 110 (43), 17450-17455 (2013).</li><li>He, L., 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+sensitive+flow+cytometry-based+cytotoxic+T-lymphocyte+assay+through+detection+of+cleaved+caspase+3+in+target+cells.">A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells.</a> J Immunol Methods. 304 (1-2), 43-59 (2005).</li><li>Sutton, V. 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. K., 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=Expression+patterns+of+NKG2A,+KIR,+and+CD57+define+a+process+of+CD56dim+NK-cell+differentiation+uncoupled+from+NK-cell+education.">Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.</a> Blood. 116 (19), 3853-3864 (2010).</li><li>Bryceson, Y. T., Ljunggren, H. G., 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=Minimal+requirement+for+induction+of+natural+cytotoxicity+and+intersection+of+activation+signals+by+inhibitory+receptors.">Minimal requirement for induction of natural cytotoxicity and intersection of activation signals by inhibitory receptors.</a> Blood. 114 (13), 2657-2666 (2009).</li><li>Cooper, 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=Human+natural+killer+cells:+a+unique+innate+immunoregulatory+role+for+the+CD56(bright)+subset.">Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset.</a> Blood. 97 (10), 3146-3151 (2001).</li><li>Fauriat, C., Long, E. O., Ljunggren, H. G., 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=Regulation+of+human+NK-cell+cytokine+and+chemokine+production+by+target+cell+recognition.">Regulation of human NK-cell cytokine and chemokine production by target cell recognition.</a> Blood. 115 (11), 2167-2176 (2010).</li><li>Postow, M. A., Callahan, M. K., Wolchok, 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=Immune+Checkpoint+Blockade+in+Cancer+Therapy.">Immune Checkpoint Blockade in Cancer Therapy.</a> J Clin Oncol. 33 (17), 1974-1982 (2015).</li><li>Glienke, W., 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=Advantages+and+applications+of+CAR-expressing+natural+killer+cells.">Advantages and applications of CAR-expressing natural killer cells.</a> Front Pharmacol. 6, 21 (2015).</li><li>Curran, K. J., Brentjens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+for+cancer+immunotherapy.">Chimeric antigen receptor T cells for cancer immunotherapy.</a> J Clin Oncol. 33 (15), 1703-1706 (2015).</li><li>Zappasodi, R., de Braud, F., Di Nicola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoma+Immunotherapy:+Current+Status.">Lymphoma Immunotherapy: Current Status.</a> Front. Immunol. 6, 448 (2015).</li><li>Laprevotte, 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=Endogenous+IL-8+acts+as+a+CD16+co-activator+for+natural+killer-mediated+anti-CD20+B+cell+depletion+in+chronic+lymphocytic+leukemia.">Endogenous IL-8 acts as a CD16 co-activator for natural killer-mediated anti-CD20 B cell depletion in chronic lymphocytic leukemia.</a> Leuk Res. 37 (4), 440-446 (2013).</li><li>Maloney, D. 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. K., 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+immunostimulatory+effect+of+lenalidomide+on+NK-cell+function+is+profoundly+inhibited+by+concurrent+dexamethasone+therapy.">The immunostimulatory effect of lenalidomide on NK-cell function is profoundly inhibited by concurrent dexamethasone therapy.</a> Blood. 117 (5), 1605-1613 (2011).</li><li>Salih, 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=The+BCR/ABL-inhibitors+imatinib,+nilotinib+and+dasatinib+differentially+affect+NK+cell+reactivity.">The BCR/ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity.</a> Int J Cancer. 127 (9), 2119-2128 (2010).</li><li>Benson, D. 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=A+phase+1+trial+of+the+anti-KIR+antibody+IPH2101+in+patients+with+relapsed/refractory+multiple+myeloma.">A phase 1 trial of the anti-KIR antibody IPH2101 in patients with relapsed/refractory multiple myeloma.</a> Blood. 120 (22), 4324-4333 (2012).</li><li>Vey, N., 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+phase+1+trial+of+the+anti-inhibitory+KIR+mAb+IPH2101+for+AML+in+complete+remission.">A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission.</a> Blood. 120 (22), 4317-4323 (2012).</li><li>Terme, M., Ullrich, E., Delahaye, N. F., Chaput, N., Zitvogel, L. <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-directed+therapies:+moving+from+unexpected+results+to+successful+strategies.">Natural killer cell-directed therapies: moving from unexpected results to successful strategies.</a> Nat Immunol. 9 (5), 486-494 (2008).</li></ol>

De beschreven werkwijze is een eenvoudige, snelle en betrouwbare benadering NK celfuncties bestuderen monsters volbloed van gezonde donoren of patiënten. Deze werkwijze biedt het grote voordeel om NK-cellen uit bloed rechtstreeks zuiveren, het vermijden van tijdrovende dichtheidsgradiënt centrifugatie, wat verplicht is voor vele andere zuiveringsmethoden 15. Bovendien, het vereist een kleinere steekproef omvang ten opzichte van de &quot;klassieke&quot; NK cel isolatie / verrijking methoden, waardoor het een...

Als onderdeel van het aangeboren immuunsysteem natural killer (NK) cellen bijdragen tot de eerste verdedigingslinie tegen virus geïnfecteerde of kwaadaardig getransformeerde cellen. Een systeem van remmende en activerende receptoren kunnen zij onderscheiden tussen gezonde en getransformeerde cellen zonder voorafgaande priming antigeen in tegenstelling tot T-cellen. Bij doelcel ontmoeting NK cellen geven de inhoud van hun cytotoxische granules (bijv perforine, granzymen) naar het immuunsysteem synaps om hun doel te doden. Bovendien, NK cellen en scheiden verschillende cytokinen (bijvoorbeeld interferon-γ: IFN-γ, tumornecrosefactor-α: TNF-α) en chemokinen (bijvoorbeeld macrofaag inflammatoir proteïne-1β: MIP-1β) bij doelcel interactie of cytokinestimulatie 1. Voldoende NK celfuncties zoals cytotoxiciteit, chemokine en cytokine productie hebben een belangrijke impact hebben op het lot van diverse disvergemakkelijkt. Leukemie patiënten vertonen verhoogde recidief als ze een defecte NK-cel profiel vertonen bij diagnose, bestaande uit gereduceerde IFN-γ productie en een verminderde expressie van het activeren van NK-cel-receptoren 2. Een snel herstel van NK-cel aantallen en functie zoals cytokineproductie bij doelcel interactie geassocieerd met een verlaagd recidief en verbeterde overleving bij patiënten die allogene stamceltransplantatie 3. Bovendien, bij aanvang van interferon therapie bij hepatitis C-virus geïnfecteerde patiënten de degranulatie aantal perifere NK cellen sterker begin responders dan bij niet-responders 4. NK-cel aantallen (&gt; 80 / ul) op dag 15 na autologe stamceltransplantatie (autoSCT) bij patiënten met lymfoom en multiple myeloom predictief voor een betere progressievrije en totale overleving 5. Bij melanoompatiënten de expressie van de T-cel immunoglobulin- en mucine-domein met daG-3 molecule (TIM-3), een immuun-regulerende eiwit op NK-cellen, correleert met ziektestadium en prognose 6. Wetenschappers hebben NK celfuncties gevolgd gedurende de laatste decennia. De eerste analyse van de NK-cel cytotoxiciteit tegen tumorcellen zonder voorafgaande priming werd aangepakt met behulp van een 51 Cr-afgifte test 7. Recenter ontwikkelde wetenschappers een niet-radioactieve methode om de cytotoxiciteit van NK cellen geëxpandeerde 8 evalueren. Cytokine en chemokine productie is herhaaldelijk geëvalueerd middels enzymgekoppelde immunosorbent assay (ELISA) technieken 9,10. Gedurende de laatste decennia deze methoden zijn aangevuld met flowcytometrie-gebaseerde testen. Het gebruik van eiwit transport remmers (bijv Brefeldine A en monensine) en cel permeabilisatie methoden in combinatie met conventionele oppervlak kleuring protocollen konden wetenschappers chemokine en cytokine productie in verschillende specifieke lymfe bestuderenocyte subsets (bijvoorbeeld T, B of NK-cellen) 11. Bovendien zijn verschillende stroomcytometrie gebaseerde bepalingen ontwikkeld om toezicht T- en NK-cel cytotoxiciteit. In 2004 beschreven Alter et al. De oppervlakte-expressie van het lysosoom-geassocieerd eiwit CD107a (Lamp1) op NK cellen na doelwitcel tegenkomen als een marker voor de degranulatie van cytotoxische granules 12. Omdat een groot aantal verschillende fluorochromen en meerkanaals flowcytometers zijn in onze dagen, is het mogelijk om gelijktijdig bewaken NK diverse celfuncties (cytotoxiciteit, cytokine en chemokine productie) in verschillende NK cel subsets. Dit wordt met name belangrijk in situaties waarin steekproefomvang beperkt is, bijvoorbeeld in biopten of bloedmonsters van patiënten met leukopenie. Wereldwijde NK celfuncties te testen, kunnen de verschillende stromingscytometrie gebaseerde testen efficiënt worden gecombineerd. Theorell et al. Gestimuleerde NK-cellen uit HEAlthy donoren met de tumor-cellijn K562 en geanalyseerd NK celdegranulatie, inside-out signaal en chemokine productie via flowcytometrie 13. Onlangs NK-cel subgroepen fenotypes en functie in tumor patiënten tijdens autoSCT werden geanalyseerd met flowcytometrie-gebaseerde testen. Er werd aangetoond dat NK-cellen konden degranuleren en cytokinen / chemokinen na tumorcel erkenning in zeer vroege tijdstippen na autoSCT 11. Hier een protocol wordt beschreven NK celfuncties na interactie evalueren tumorcellen waaronder degranulatie capaciteit chemokine en cytokine productie met een flow cytometrie-gebaseerde test die het mogelijk maakt om NK celfuncties bij verschillende subgroepen kan waarnemen.

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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>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		<tr height="21">
			<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>
		</tr>
		<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.

<html> De gating strategie voor het analyseren van de degranulatie, cytokine en chemokine productie van de gehele NK celpopulatie en drie verschillende subsets van NK cellen zijn aangegeven in figuur 1. Representatieve resultaten van een gezonde donor zijn weergegeven in figuur 2. NK-cellen zonder prikkel geproduceerd noch IFN-γ of MIP-1β en niet CD107a expressie op hun oppervlak (Fig...

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

Een eenvoudige en betrouwbare werkwijze is beschreven om een ​​set van NK celfuncties zoals degranulatie cytokine en chemokine productie in verschillende NK cel subsets te analyseren.

Flowcytometrie-gebaseerde test voor het toezicht op NK celfuncties

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

Research

JoVE Journal

Immunology and Infection

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

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

Monitoring Dendritische cel-migratie met behulp van<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

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 ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Het analyseren van de functies van mastcellen in Vivo met behulp van ' MastCell Knock-in' Muizen

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

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

Isolatie en Flow cytometrische analyse van de immuuncellen van de ischemische hersenen van muizen

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

Fluorescentie-geactiveerde Cell Sorting voor Zuivering van plasmacytoïde dendritische cellen van de muis Bone Marrow

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

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

Het beoordelen van het netvlies Microgliale fagocytosefunctietesten<em&gt; In Vivo</em&gt; Met behulp van een flowcytometrie-gebaseerde test

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

Dextran verbetert de efficiëntie van de lentivirale transductie van lymfkliertest en menselijke primaire NK-cellen

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

Een stroom Cytometry gebaseerde cytotoxiciteit Assay voor de beoordeling van menselijke NK cel activiteit

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

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

Een eenvoudige Stroom Cytometry gebaseerd Assay om te bepalen In Vitro antilichaam afhankelijk verbetering van Dengue Virus met behulp van de herstellende Serum Zika-Virus

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

Multicolor Stroom Cytometry gebaseerde kwantificering van Mitochondria en lysosomen in T cellen

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