Name: flow cytometric analysis of natural killer cell lytic activity in human whole blood
Uploaded: 2017-03-17T12:30:00
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.

NOTA: Todos los procedimientos de recolección de sangre se llevaron a cabo de acuerdo con las directrices establecidas por la Universidad Estatal de los Apalaches (ASU) Junta de Revisión Institucional (IRB). 1. Extracción de sangre entera <ol><li> Tiene un flebotomista certificado extraer la sangre según las directrices de la Organización Mundial de la Salud. </li><li> Extraer la sangre en un tubo de recogida de sangre de 4 ml que contenía ácido etilendiaminotetraacético Di-Potasio...

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El método descrito en este estudio mide directamente la actividad específica de las células NK de un individuo en respuesta a estímulos (en este caso particular, el ejercicio prolongado). Típicamente, las células NK están aisladas de la propia sangre usando gradientes de densidad o clasificación de células mediante el uso de una combinación de marcadores. Aunque se utilizan ampliamente estos métodos, que tienen muchos inconvenientes: son mucho tiempo, implican múltiples manipulaciones, y son en gran medida d...

Las células asesinas naturales son un elemento esencial del sistema inmune innato. Mientras que están muy regulados, que tienen la capacidad de reconocer y eliminar las células anormales a través del contacto de célula a célula y sin activación previa 1. Como tales, forman una línea rápida de defensa contra las infecciones. El ejercicio, especialmente intenso, se ha demostrado que deprimen de forma transitoria el sistema inmune 2, 3, 4, 5. Las células NK son particularmente propensos a este efecto 4, 6, 7, creando una ventana de aumento de la sensibilidad a la enfermedad. Por lo tanto, el estudio de las intervenciones antes, durante o después del ejercicio intenso, con el objetivo de reducir su impacto sobre la función de las células NK es de particular interés para el bienestar de los atletas después de los concursos. 8, en torno a 1% del compartimiento de glóbulos blancos; 2) Las células NK son muy sensibles al estrés y se basan en la exposición constante a las condiciones fisiológicas para seguir siendo viable y estable durante la experimentación; y 3) ensayos estándar para determinar la citotoxicidad de células NK, tales como gradientes de Ficoll 9 y liberan los ensayos 10, son poco fiables e inconsistente. La variabilidad inherente de las muestras de humanos sólo agravan estos problemas. muestras humanas frescas recogidas durante las intervenciones son bastante regulado y difíciles de conseguir, al menos en comparación con muestras de animales o líneas celulares inmortalizadas. Esto reduce las oportunidades para repetir los experimentos o añadir participantes a la cohorte del estudio para llegar a los umbrales estadísticos significativos. En conjunto, estas cuestiones apoyan la necesidad de un protocolo optimizado que permite for tanto de alto rendimiento y un análisis de alta fiabilidad de la actividad lítica de las células NK en muestras humanas. Hemos establecido un flujo de trabajo que acorta el tiempo necesario para identificar, aislar y células NK prueba de la sangre entera humana y reducir al mínimo la exposición a factores externos. El método optimiza el uso de dos instrumentos, un clasificador basado en magnética celular y un flujo de imágenes citómetro, y un, T optimizado ensayo específico: relación E para permitir la detección de las disminuciones o incrementos de la citotoxicidad de células NK.

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			<td height="21" style="height:21px;width:371px;">K-562 lymphoblasts</td>
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			<td height="21" style="height:21px;width:371px;">Propridium Iodide Staining Solution</td>
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			<td height="21" style="height:21px;width:371px;">Vybranto DiO cell-labeling solution</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Pro Separator</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Running Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Washing Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
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			<td height="21" style="height:21px;width:371px;">autoMACS Columns</td>
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			<td height="21" style="height:21px;">Whole Blood CD56 MicroBeads, human</td>
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			<td height="21" style="height:21px;">ImageStream X Mark II Imaging Flow Cytometer</td>
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			<td style="width:136px;">1.3704</td>
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			<td height="21" style="height:21px;">D-PBS (Sheath fluid)</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;">BSS-1006-B (1X)</td>
			<td>No calcium or magnesium</td>
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			<td height="21" style="height:21px;">INSPIRE Software</td>
			<td style="width:183px;">EMD Millipore</td>
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flow cytometric analysis of natural killer cell lytic activity in human whole blood

la citotoxicidad de células NK es una medida ampliamente utilizada para determinar el efecto de la intervención exterior en función de las células NK. Sin embargo, la precisión y reproducibilidad de este ensayo pueden considerarse inestable, ya sea debido a errores del usuario o debido a la sensibilidad de las células NK a la manipulación experimental. Para eliminar estos problemas, un flujo de trabajo que les reduce al mínimo fue establecido y se presenta aquí. Para ilustrar, se obtuvieron muestras de sangre, en diversos momentos, desde corredores (n = 4) que fueron sometidos a una intensa sesión de ejercicio. En primer lugar, las células NK se identifican de forma simultánea y aislados mediante el etiquetado CD56 y la clasificación basada en magnético, directamente de la sangre entera y de tan poco como un mililitro. Las células NK según se retiran de cualquier anticuerpo reactivo o de tapado. Se puede contar con el fin de establecer un recuento de células NK precisa por mililitro de sangre. En segundo lugar, las células NK según (efectores células o E) se pueden mezclar con 3,3&#39;-Diotadecyloxacarbocyanine perclorato (DIO) etiquetada células K562 (células diana o T) en un ensayo-óptima 1: 5 T: relación de E, y se analizó usando un citómetro de flujo de imágenes que permite la visualización de cada evento y la eliminación de cualquier falso positivo o falsos negativos (tales como dobletes o células efectoras). Este flujo de trabajo se puede completar en aproximadamente 4 h, y permite obtener resultados muy estables incluso cuando se trabaja con muestras humanas. Cuando esté disponible, los equipos de investigación pueden probar varias intervenciones experimentales en sujetos humanos, y comparar las mediciones a través de varios puntos de tiempo sin poner en peligro la integridad de los datos.

Determinación del número de células NK Se midió el efecto de la pesada que se ejecuta en el recuento de células NK en la sangre total, utilizando el protocolo de ejercicio descrito en la figura 2. Las muestras de sangre fueron extraídas antes del ejercicio, inmediatamente después del ejercicio, 1,5 horas después del ejercicio, y, finalmente, 24 y 48 h después de la extracción de sangre inicial. La concentración de las células NK por mililitro de sangre...

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.

Análisis de citometría de flujo de las células asesinas naturales lítico Actividad en sangre entera humana

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

The overall goal of this experiment is to determine NK cell count and cytotoxic activation levels in a human whole blood sample in a fast, reliable and stable manner. This method can help answer questions in the immunological fields, such as, What are the effects of outside interventions on the bioactivity of NK cells? The main advantage of this technique is that it is fast and accurate, While still providing reproducible results across experiments.To isolate NK cells from human whole blood, first place 15 milliliter tubes labeled Whole blood sample, Negative Fraction and Positive Fraction into the appropriate positions in the cell separator tube rack. When all of the tubes are loaded, insert the separator rack onto the mini-sampler for auto labeling, and select Reagent on the menu, to highlight the position where the vial will be placed on the rack. Select Read Reagent to activate the 2D code reader and hold the appropriate reagent vial in front of the 2D code reader at an angle 0.5 to 2.5 centimeters from the code reader cover.Place the read vial into the appropriate separator rack position. Next, under the Separation tab, highlight the desired positions. Then open the Labeling submenu and select an auto-labeling program.Assign the cell separation reagent microbeads to rack positions 1, 2, 3 and 4. And select the Posselwb separaton program. Select the Rinse Wash program.Enter 1500 microliters for the Sample Volume under the Volume sub menu, and press Enter. Then click Run to begin the cell separation. Clicking okay to confirm that enough buffer is available for processing all of the samples.To prepare a Cytotoxicity Assay sample, first, label 1.5 milliliter tubes for each sample and/or participant as appropriate, and pipe out the desired ratio of NK cells and DIO labeled K562 cells into each tube. Collect the cells by centrifugation, and carefully remove the supernatant without disturbing the cell pellet. Next, resuspend the cell mixture in 500 microliters of incomplete NK cell medium, and label the cells in each tube with 5 microliters of propidium iodide.Collect the cells via another centrifugation, and incubate the cull culture at 37 degrees celsius for two hours. At the end of the incubation, centrifuge the cells under the same centrifuge conditions, and resuspend the pellet in 25 microliters of fresh incomplete NK cell culture medium. To analyze the cells by Flow Cytometry, first, click the start up button in the imaging flow cytometer software to flush the system.Next, click on the scatterplot button to create four scatterplots, and load the double positive control tube. Using the double positive control sample, determine the desired intensity for the 405 milliwatt laser, so that the detector is not overloaded, and set the imager to acquire the appropriate number of cell counts. Setting the 405 milliwatt laser at an intensity that is suitable for the detection of both DIO and PI will require experience with both dyes and the detection range of the flow cytometer.After the acquisition of the double positive control sample finishes, load the DIO only control sample, select the 40X objective, and turn off the 642 milliwatt laser and the Brightfield channel. Breed the appropriate number of cells and repeat the acquisition for the propidium iodide only sample. When all of the control samples have been run, turn the 405 milliwatt laser and Brightfield channel, back on, and click Set Intensity.Then, under the File Acquisition tab, enter a custom file name, and select a folder for saving the data files. Next, enter the number of cells to acquire. Load the first experimental sample tube and click the Acquire button.When all of the samples have been run, open the imaging flow cytometer analysis software application, and open a single experimental RIF file. Under the compensation tab, select Create New Matrix. The software will prompt for the selection of the single color files, and merge them to create a matrix file to be selected for applying the channel compensation.Use the Building Blocks function to set up the appropriate dot plots. Then click on the statistics function to access the cell numbers in each gate. In this representative experiment, blood samples were drawn as indicated and the concentration of NK cells per milliliter of whole blood was measured for each runner, and on average for each time point.Three out of four runners presented a similar slight increase in NK cell count after exercise. Immediately followed by a sharp decrease, before slowly returning to normal, 24 to 48 hours after the end of the event. On average the NK cell count decreased 1.5 hours after exercise, but returned to near normal levels after 24 hours.After calculating the cell count, the cytotoxic activity of the NK cells was immediately analyzed as just demonstrated, with an average decrease in NK cell cytotoxicity immediately, and 1.5 hours after the termination of the exercise event, that increased significantly by 24 hours post exercise. The NK cytotoxicity remained high compared to the pre-exercise levels, albeit nonsignificantly. Once mastered, this technique can be completed in about four hours when performed properly.After watching this video, you should have a good understanding of how to obtain and characterize NK cells count and cytotoxic activity from human blood samples in a reproducible hydropic manner.

Research

JoVE Journal

Immunology and Infection

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Super-resolución de imagen de la célula Natural Killer inmunológica Synapse en un apoyada-Glass planar bicapa lipídica

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Investigación

Inmunología e Infección

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Imaging Whole-animal y de citometría de flujo Las técnicas para T CD8 + respuestas celulares Análisis de antígeno-específicos después de nanopartículas Vacunación

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

El aislamiento y la citometría de flujo análisis de las células mononucleares de sangre periférica Glioma infiltrantes

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Una citometría de flujo simple método para medir la captación de glucosa y la expresión del transportador de la glucosa por monocitos subpoblaciones en sangre entera

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Análisis de citometría de flujo de partículas unida a Bet v 1 de alérgenos en el MP10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Aislamiento y Análisis de citometría de flujo, de Human endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Análisis unicelular de Immunophenotype y producción de citoquinas en sangre periférica por citometría de masas

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Caracterización de subconjuntos de monocitos humanos de sangre análisis de citometría de flujo

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...