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.

Denne undersøgelse blev udført i overensstemmelse med anbefalingerne fra den lokale etiske udvalg fra University of Frankfurt. 1. Dyrkning af K562-celler <ol><li> Kultur K562-celler i R10 medier (RPMI1640 med glutamin-medium, 1% penicillin / streptomycin, 10% føtalt kalveserum) ved en densitet på 0,5-1 x 10 6 celler pr ml i en cellekultur kolbe ved 37 ° C og 5% CO 2. </li><li> Harvest K562 Cells 24 timers Før starten af ​​et nyt eksperiment. <ol><li> cellekulturen kolbe indehold...

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Den beskrevne fremgangsmåde er en let, hurtig og pålidelig metode til at studere NK celle funktioner fra prøver af fuldblod fra raske donorer eller patienter. Denne fremgangsmåde giver den store fordel at direkte oprense NK-celler fra fuldblod, undgå tidskrævende densitetsgradientcentrifugering, som er obligatorisk for mange andre oprensningsmetoder 15. Desuden kræver det en mindre stikprøve i forhold til &quot;klassiske&quot; NK-celle isolation / berigelse metoder, hvilket gør det til et egnet alter...

Som en del af det medfødte immunsystem naturlige dræberceller (NK-celler) bidrager til den første linje i forsvar mod virus-inficerede eller malignt transformerede celler. Et system af inhiberende og aktiverende receptorer gør dem i stand til at skelne mellem raske og transformerede celler uden forudgående antigenpriming i modsætning til T-celler. Ved target celle støder NK-celler frigiver indholdet af deres cytotoksiske granulat (f.eks perforin, granzymer) i immunsystemet synapsen til at dræbe deres mål. Desuden NK-celler producerer og udskiller forskellige slags cytokiner (fx interferon-y: IFN-γ; tumornekrosefaktor-α: TNF-α) og chemokiner (f.eks makrofag inflammatorisk protein-1β: MIP-1) efter målcelle interaktion eller cytokinstimulering 1. Tilstrækkelige NK celle funktioner såsom cytotoksicitet, chemokin og cytokinproduktion har en betydelig indvirkning på den skæbne diverse disletter. Leukæmi patienter viser øget tilbagefald, hvis de udviser en defekt NK-celle-profil ved diagnose bestående af reduceret IFN-γ produktion og reduceret ekspression af aktiverende NK celle receptorer 2. En tidlig genopretning af NK celle numre og funktion, herunder cytokinproduktion på target celle interaktion er forbundet med en reduceret tilbagefald og forbedret overlevelse hos patienter i allogen stamcelletransplantation 3. Desuden ved initiering af interferonbehandling i virus-inficerede patienter hepatitis C i degranulering kapacitet af perifere NK-celler er stærkere i de tidlige respondenter end hos ikke-responders 4. NK celle tal (&gt; 80 / ul) på dag 15 efter autolog stamcelletransplantation (autoSCT) hos patienter, der lider af lymfom eller myelomatose er prædiktive for en forbedret progressionsfri og samlet overlevelse fem. I melanompatienter ekspressionen af ​​T-celle immunoglobulin- og mucin-domæne-containing molekyle-3 (TIM-3), en immun-regulatorisk protein på NK-celler, korrelerer med sygdom fase og prognose 6. Forskere har overvåget NK cellefunktioner gennem de seneste årtier. Den indledende analyse af NK-celle cytotoksicitet mod tumorceller uden forudgående priming blev behandlet ved hjælp af en 51 Cr-release assay 7. For nylig har forskere udviklet en ikke-radioaktiv fremgangsmåde til evaluering af cytotoksicitet af ekspanderede NK-celler 8. Cytokin og chemokin produktion er ofte blevet evalueret ved brug enzymmaerket assay (ELISA) teknikker 9,10. I de sidste årtier disse metoder er blevet suppleret med flowcytometri-baserede assays. Brugen af protein transport hæmmere (f.eks Brefeldin A og monensin) og celle permeabilisering metoder i kombination med konventionel overflade farvningsprotokoller har aktiveret forskere til at studere chemokin og cytokinproduktion i forskellige specifikke lymfeknuderocyte delmængder (fx T, B eller NK-celler) 11. Desuden har forskellige flowcytometri assays blevet udviklet til at overvåge T og NK-cellers cytotoksicitet. I 2004 beskrev Alter et al. Overfladeekspressionen af lysosomet-associeret protein CD107a (Lamp1) på NK-celler ved målcelle støder som markør for degranulering af cytotoksiske granuler 12. Da en lang række forskellige fluorochromer og flerkanals-flowcytometre er tilgængelige i vore dage er det blevet muligt samtidigt at overvåge forskellige NK cellefunktioner (cytotoksicitet, cytokinreceptorer og kemokinproduktion) i forskellige NK-celle delmængder. Dette bliver især vigtigt i situationer, hvor stikprøve størrelse er begrænset, f.eks i biopsier eller blodprøver fra patienter, der lider leukopeni. For at teste globale NK cellefunktioner, kan de forskellige flowcytometri assays effektivt kombineres. Theorell et al. Stimulerede NK-celler fra healthy donorer med tumoren K562 og analyseret NK-celle degranulering, inside-out signal og chemokin produktion via flowcytometri 13. Senest NK-celle-undergrupper, fænotyper og funktioner i tumor patienter under autoSCT blev analyseret ved anvendelse af flowcytometri-baserede assays. Det blev påvist, at NK-celler var i stand til at degranulere og producere cytokiner / chemokiner upon tumorcelle anerkendelse på meget tidlige tidspunkter efter autoSCT 11. Her en protokol er beskrevet for at evaluere NK celle funktioner ved interaktion med tumorceller inklusiv degranulering kapacitet, chemokin og cytokinproduktion ved anvendelse af en flow-cytometri-baseret assay, der gør det muligt at overvåge NK celle funktioner i forskellige undersæt samtidigt.

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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>
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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>
		</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> Gating strategi til analyse af degranulering, cytokin og chemokin fremstillingen af hele NK-celle population og tre forskellige NK-celle delmængder er illustreret i figur 1. Repræsentative resultater af en rask donor er illustreret i figur 2. NK-celler uden stimulus produceret hverken IFN-γ eller MIP-1β og udtrykte ikke CD107a på deres overflade (figur 2A). I modsæ...

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

En enkel og pålidelig fremgangsmåde er beskrevet her for at analysere et sæt af NK cellefunktioner såsom degranulering, cytokin og chemokin produktion inden for forskellige NK-celle delmængder.

Flowcytometri-baserede assay for Overvågning af NK cellefunktioner

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

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

Cell-baserede Flowcytometri Assay til måling cytotoksisk aktivitet

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

Analyse af mastcellernes funktioner i Vivo Ved hjælp af 'Mast Cell Knock-in' Mus

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

Isolering og flowcytometrisk analyse af immunceller fra iskæmisk Mouse Brain

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

Fluorescens-aktiveret cellesortering til oprensning af Plasmacytoide dendritiske celler fra knoglemarven i mus

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

Vurdering Retinal mikrogliaceller fagocytotisk funktion<em&gt; In vivo</em&gt; Brug en flowcytometri-baseret assay

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 effektiviserer Lentiviral transduktion af Murine og menneskelige primære NK-celler

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

En Flow flowcytometri-baserede cytotoksicitet analyse til vurdering af menneskers NK celleaktivitet

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

En simpel flowcytometri baseret analyse for at bestemme In Vitro antistof afhængig af forbedring af Dengue Virus ved hjælp af Zika Virus rekreationshjem Serum

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

Flerfarvet Flow flowcytometri-baserede kvantificering af mitokondrier og lysosomer i T celler

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