Name: flow cytometric analysis of natural killer cell lytic activity in human whole blood
Uploaded: 2017-03-17T12:30:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood at JoVE.com

This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2100-68003-30395 from the USDA National Institute of Food and Agriculture.

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Further observations. Methylcellulose, dextran, and ficoll as erythrocyteaggregating agents.</a> Scand J Clin Lab Invest Suppl. 97, 31-50 (1968).</li><li>McMillan, R., Scott, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+labeling+with+51-Chromium.+I.+Technic+and+results+in+normal+subjects.">Leukocyte labeling with 51-Chromium. I. Technic and results in normal subjects.</a> Blood. 32 (5), 738-754 (1968).</li><li>Berk, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+long+endurance+running+on+natural+killer+cells+in+marathoners.">The effect of long endurance running on natural killer cells in marathoners.</a> Med Sci Sports Exerc. 22 (2), 207-212 (1990).</li><li>McAnulty, L. 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The authors have nothing to disclose.

本研究に記載された方法は、直接的（この特定の場合、長時間の運動で）刺激に応答して個々のNK細胞の特異的活性を測定します。典型的に、NK細胞は、マーカーの組み合わせを使用して選別密度勾配または細胞を使用して1つの血液から単離されます。これらの方法が広く使用されているが、それらは多くの欠点を持っている：彼らは、時間がかかり、複数の操作を伴い、かつ重くユーザー?...

ナチュラルキラー細胞は、自然免疫系の重要な構成要素です。それらは非常に規制されているが、それらは認識し、細胞間接触を介して前活性化1せずに異常な細胞を除去する能力を有します。そのようなものとして、それらは、感染に対する防御の迅速なラインを形成します。特に激しい運動は、一過性免疫系2、3、4、5を抑制することが示されています。 NK細胞は、効果的に疾患に高感度のウィンドウを作成し、この効果4、 図6、 図7を特に受けやすいです。したがって、前の介入の研究は、NK細胞機能への影響を削減するという目標との間または後の激しい運動は、運動選手のポスト競技の幸福のために特に重要です。 1％、8まばらです。 2）NK細胞はストレスに非常に敏感であり、実験中に生存可能で安定した状態を維持するために生理学的条件に一定の暴露に依存しています。 3）標準的なアッセイは、フィコール勾配9として、NK細胞の細胞毒性を決定し、アッセイ10を解放するために、信頼できないと矛盾しています。ヒト試料の固有の変動は、これらの問題を配合します。介入の間に収集され、新鮮なヒトサンプルは、少なくとも動物サンプルまたは不死化細胞株と比較した場合、入手がかなり規制が困難です。これは、実験を繰り返したり、統計的な有意しきい値に到達するために研究コホートに参加者を追加する機会を減らすことができます。まとめると、これらの問題はfoを可能にする合理化されたプロトコルの必要性をサポートrの両方のハイスループットおよびヒト試料中のNK細胞溶解活性の高い信頼性解析。 無関係な要因への曝露を最小限に抑えながら、我々は、ヒト全血から識別するために必要な時間を短縮し、ワークフロー、隔離し、テストNK細胞を確立しました。減少またはNK細胞の細胞毒性の増加の検出を可能にするE比：方法は2楽器、磁気ベースのセルソーターとイメージングフローサイトメーター、およびアッセイ特異的な、最適化されたTの使用を最適化します。

<table><tbody><tr><td>K-562 lymphoblasts</td><td>ATCC</td><td>CCL-243</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Media</td><td>ATCC</td><td>30-2005</td><td>High glucose, with L-Glutamine, with HEPES, Sterile-filtered</td></tr><tr><td>Alpha Minimum Essential medium</td><td>ATCC</td><td>CRL-2407</td><td>Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate</td></tr><tr><td>Trypan Blue Solution 0.4%</td><td>Amresco</td><td>K940-100ML</td><td>Tissue culture grade</td></tr><tr><td>Propridium Iodide Staining Solution</td><td>BD Pharmingen</td><td>51-66211E</td><td></td></tr><tr><td>Vybranto DiO cell-labeling solution</td><td>Vybranto</td><td>V-22886</td><td></td></tr><tr><td>autoMACS Pro Separator</td><td>Miltenyi Biotec</td><td>130-092-545</td><td></td></tr><tr><td>autoMACS Running Buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>autoMACS Washing Buffer</td><td>Miltenyi Biotec</td><td>130-092-987</td><td></td></tr><tr><td>autoMACS Columns</td><td>Miltenyi Biotec</td><td>130-021-101</td><td></td></tr><tr><td>Whole Blood CD56 MicroBeads, human</td><td>Miltenyi Biotec</td><td>130-090-875</td><td></td></tr><tr><td>ImageStream X Mark II Imaging Flow Cytometer</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Speedbeads</td><td>Amnis Corporation</td><td>400030</td><td></td></tr><tr><td>0.4-0.7% Hypochlorite (Sterilizer)</td><td>VWR</td><td>JT9416-1</td><td></td></tr><tr><td>Coulter Clenz</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>70% Isopropanol (Debubbler)</td><td>EMD Millipore</td><td>1.3704</td><td></td></tr><tr><td>D-PBS (Sheath fluid)</td><td>EMD Millipore</td><td>BSS-1006-B (1X)</td><td>No calcium or magnesium</td></tr><tr><td>INSPIRE Software</td><td>EMD Millipore</td><td></td><td>Version Mark II, September 2013</td></tr><tr><td>Ideas Application Software</td><td>EMD Millipore</td><td></td><td>Version 6.1, July 2014</td></tr></tbody></table>

flow cytometric analysis of natural killer cell lytic activity in human whole blood

NK細胞の細胞傷害性は、NK細胞機能の外部の介入の効果を決定するために広く使用される尺度です。しかし、このアッセイの精度および再現性があるため、ユーザのエラーのため、または実験的操作にNK細胞の感受性のいずれか、不安定と考えることができます。これらの問題を解消するために、最小限にそれらを減少させるワークフローを確立し、ここで提示されています。例示するために、我々は、運動の激しい試合に提出されたランナー（N = 4）から、様々な時点で、血液サンプルを得ました。まず、NK細胞は、同時にCD56のタグ付けと磁気ベースの選別を経て、直接全血からと1ミリリットル限り少ないから同定および分離されています。ソートされたNK細胞は、任意の試薬またはキャッピング抗体から除去されます。これらは、血液1ミリリットル当たりの正確なNK細胞数を確立するために計数することができます。第二に、ソートされたNK細胞（エフェクター細胞またはE）は、3,3&#39;- Diotadeと混合することができます5 T：cyloxacarbocyanine過塩素酸（DIO）をアッセイ最適1でK562細胞（標的細胞またはT）をタグ付けされたE比、および各イベントの可視化および任意の偽陽性の排除を可能にするイメージングフローサイトメーターを用いて分析しましたまたは（例えばダブレットまたはエフェクター細胞など）偽陰性。このワークフローは、約4時間で完了し、さらにはヒト試料での作業は非常に安定した結果を可能にすることができます。ご利用の際は、研究チームは、ヒト対象にいくつかの実験介入をテストし、データの整合性を損なうことなく、いくつかの時点全体の測定値を比較することができます。

NK細胞数の決意 全血におけるNK細胞数の激しい走行の効果は、図2に記載の運動プロトコルを用いて、測定されました。血液サンプルは直ちに運動、運動後1.5時間、および初期採血後、最終的に24および48時間後に、運動前に描かれました。全血1ミリリットル当たりNK細胞の濃度は、各ランナー（ 図3A）および各時点の平均（ 図3B）</s...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood at JoVE.com

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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Research

JoVE Journal

Immunology and Infection

12.4K ビュー.  North Carolina A&T State University, North Carolina Research Campus. This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ビデオ: Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

免疫・感染学

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

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

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

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

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.

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

NK細胞の機能を監視するためのサイトメトリーに基づくアッセイを流します

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

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

ヒト NK 細胞活性の評価の流れのフローサイトメトリーを用いた細胞毒性の試金

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

治療抗体の存在下におけるFcγRIIIa関与によるヒトナチュラルキラー細胞事象の評価

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

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.

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.

ヒトナチュラルキラー細胞代謝の解析

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 ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

<meta charset="utf8"></head><body>ヒトナチュラルキラー細胞受容体-リガンドレパートリーのプロファイリング

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

ヒトナチュラルキラー細胞受容体-リガンドレパートリーのプロファイリング

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

肝腫瘍細胞の文脈における自然キラー細胞媒介性細胞毒性と遊性の測定

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

医学

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Source: McBride, J. E., et al. <a href="https://app.jove.com/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

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