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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The authors have nothing to disclose.

השיטה המתוארת במחקר זה ישירות מודדת את הפעילות הספציפית של תאי NK של פרט בתגובה לגירויים (במקרה המסוים הזה, פעילות הגופנית ממושכת). בדרך כלל, תאי NK מבודדים מהדם של אחד על ידי מפלי צפיפות או מיון התא באמצעות שילוב של סמנים. בעוד שיטות אלה נמצאים בשימוש נרחב, יש להם חסרונו?...

<html> תאי הרג טבעיים הם מרכיב חיוני של מערכת החיסון המולדת. למרות שהם מוסדרים מאוד, יש להם את היכולת לזהות ולסלק תאים חריגים באמצעות מגע תא אל תא וללא לפני ההפעלה 1. ככזה, הם יוצרים קו מהיר של הגנה מפני זיהומים. אינטנסיבי תרגיל, במיוחד, מוכיח לדכא את המערכת החיסונית 2 זמני, 3, 4, 5. תאי NK הם רגישים במיוחד את האפקט הזה 4, 6, 7, ובכך נפתח חלון של רגישות משופרת באופן יעיל למחלות. לפיכך, במחקר של התערבויות לפני, במהלך או אחרי פעילות גופנית אינטנסיבית במטרה להפחית את השפעתה על תפקוד תא NK הוא בעלת עניין מיוחד עבור רווחתם של ספורטאים-תחרויות פוסט. = &quot;jove_content&quot;&gt; עם זאת, המחקר של התערבויות כאלה מסתבך בשל גורמים רבים: 1) תאי NK הם דלילים 8, בסביבות 1% של תא תא דם לבן; 2) תאי NK רגישים מאוד ללחץ ולהסתמך על חשיפה מתמדת בתנאים פיסיולוגיים להישאר קיימא ויציב במהלך ניסויים; מבחני תקן 3) לקבוע cytotoxicity תא NK, כגון הדרגתי Ficoll 9 ולשחרר מבחנים 10, הם אמינים ובלתי עקביים. ההשתנות הטבועה של דגימות אנושיות רק תחריף את הנושאים הללו. דגימות אנושיות טריות שנאספו במהלך התערבויות למדי מוסדרות וקשים להשיג, לפחות בהשוואת דגימות מן החי או שורות תאים הונצחו. פעולה זו מפחיתה הזדמנויות לשחזר ניסויים או להוספת משתתפים עוקבת המחקר להגיע ספי סטטיסטיים מובהקים. ביחד, הנושאים הללו תומכים בצורך פרוטוקול יעיל המאפשר FOr היא תפוקה גבוהה ניתוח אמינות גבוהה של פעילות ממס תא NK בדגימות אדם. הקמנו עבודה המקצרת את הזמן הדרוש כדי לזהות, לבודד תאי NK מבחן מהדם האנושי כולו תוך מזעור החשיפה גורמים חיצוניים. השיטה מייעלת את השימוש בשני מכשירים, סדרן תא מגנטי מבוסס וזרימת הדמית cytometer, וחולצת assay הספציפי, אופטימיזציה: יחס E כדי לאפשר זיהוי של ירידות או עליות של cytotoxicity תא NK.

<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

cytotoxicity תא NK הוא מדד בשימוש נרחב כדי לקבוע את ההשפעה של התערבות חיצונית על תפקוד תאי NK. עם זאת, את הדיוק ואת השחזור של assay זה יכולים להיחשב יציב, אם בשל טעויות של המשתמש או בגלל הרגישות של תאי NK כדי מניפולציה ניסויית. כדי למנוע בעיות אלה, עבודה המפחיתה אותם למינימום הוקמה ומוצגת כאן. לשם המחשה, השגנו דגימות דם, בנקודות זמן שונות, מן הרצים (n = 4) שהוגשו התקף אינטנסיבי של פעילות גופנית. ראשית, תאי NK מזוהים בו זמנית ומבודד באמצעות תיוג CD56 ומיון מגנטי מבוסס, ישירות דם שלם מ קטנה כמו למיליליטר אחד. תאי NK המסודרים יוסרו מכל נוגדנים מגיבים או מכסה. הם ניתן לספור על מנת להקים ספירת תאי NK מדויקת למיליליטר דם. שנית, תאי NK המסודרים (תאי effectors או E) ניתן לערבב עם 3,3&#39;-Diotadecyloxacarbocyanine פרכלורט (DIO) מתויג K562 תאים (תאים היעד או T) לעבר assay-אופטימלית 1: 5 T: יחס E, ונותחו באמצעות cytometer זרימה הדמיה המאפשרת הדמיה של כל אירוע חיסול כל חיובי כוזב או שלילי שגוי (כגון כפילויות או תאי מפעיל). עבודה זו יכולה להסתכם בערך 4 שעות, ומאפשרת תוצאות יציבות מאוד גם כאשר עובדים עם דגימות אנושיות. תאריך הכניסה, צוותות מחקר יכול לבדוק התערבויות ניסיון כמה בבני אדם, ולהשוות מדידות על פני מספר נקודות זמן מבלי להתפשר על השלמות של הנתונים.

קביעת ספירת תאי NK ההשפעה של ריצה כבד על ספירת תאי NK בדם כולו נמדדה, באמצעות פרוטוקול התרגיל כמתואר באיור 2. דגימות דם נלקחו לפני האימון, מיד לאחר האימון, 1.5 שעות לאחר פעילות גופנית, ולבסוף 24 ו -48 שעות לאחר לקיחת הדם הראשונית. ריכוז תאי NK למי?...

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.

ניתוח תזרים Cytometric של תא הטבעי Killer ממס פעילות דם מלא אנוש

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

flow-cytometric-analysis-natural-killer-cell-lytic-activity-human

Research

JoVE Journal

Immunology and Infection

12.4K Views.  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.

Video: 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.

הכנה ושימוש של HIV-1 CD4 ראשי מאשרום + T-תאים כמו תאים היעד של טבע Cell מבחני Killer ציטוטוקסיות

 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 ...

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.

Flow Cytometry מבוסס Assay עבור הניטור של פונקציות תא NK

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.

וזמינותו Cytotoxicity מבוססי Cytometry זרימה להערכה של פעילות תאי NK אנושי

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.

הערכה של אירועי תאי רוצח טבעי אנושי המונעים על ידי מעורבות FcγRIIIa בנוכחות נוגדנים טיפוליים

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.

ניתוח של חילוף החומרים של תאי הרוצח הטבעי האנושי

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>פרופיל של רפרטואר קולטני תאי הרג טבעיים אנושיים-ליגנד

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.

פרופיל של רפרטואר קולטני תאי הרג טבעיים אנושיים-ליגנד

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.

מדידה של הרוצח הטבעי Cell-בתיווך ציטורעילות והגירה בהקשר של תאים סרטניים בכבד

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 ...

Medicine

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.

ניתוח תזרים Cytometric של תא הטבעי Killer ממס פעילות דם מלא אנוש

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