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.

MERK: Alle blod samling prosedyrer ble utført i samsvar med retningslinjene fastsatt av Appalachian State University (ASU) Institutional Review Board (IRB). 1. Hele Blodprøvetaking <ol><li> Ha en sertifisert phlebotomist trekke blod i henhold til Verdens helseorganisasjons retningslinjer. </li><li> Trekke blod inn i en 4 ml blod samling rør som inneholder Di-Kalium Ethylenediaminetetraacetic syre (K 2 EDTA). Vend blod samling rør i henhold til produsentens instruksjoner. Hol...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marathon+training+and+immune+function.">Marathon training and immune function.</a> Sports Med. 37 (4-5), 412-415 (2007).</li><li>Simpson, R. J., Kunz, H., Agha, N., Graff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+and+the+Regulation+of+Immune+Functions.">Exercise and the Regulation of Immune Functions.</a> Prog Mol Biol Transl Sci. 135, 355-380 (2015).</li><li>Walsh, N. 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=Position+statement.+Part+one:+Immune+function+and+exercise.">Position statement. Part one: Immune function and exercise.</a> Exerc Immunol Rev. 17, 6-63 (2011).</li><li>Timmons, B. 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Metoden som beskrives i denne undersøkelsen måler direkte den spesifikke aktivitet av et individs NK-celler som respons på stimuli (i dette spesielle tilfellet, langvarig trening). Vanligvis er NK-celler isolert fra en blod ved hjelp av tetthetsgradienter eller cellesortering ved hjelp av en kombinasjon av markører. Selv om disse metodene er mye brukt, har de mange ulemper: de er tidkrevende, innebære flere manipuleringer, og er sterkt avhengig av brukeren. Som et resultat blir unødig stress plassert på de isoler...

Natural killer celler er en vesentlig del av det medfødte immunsystemet. Selv om de er meget regulert, har de evnen til å gjenkjenne og eliminere unormale celler via celle-til-celle-kontakt og uten forutgående aktivering 1. Som sådan, de danner en rask linje i forsvaret mot infeksjoner. Mosjon, særlig intens, har vist seg å transient hemmer immunsystemet 2, 3, 4, 5. NK-celler er særlig utsatt for denne effekten 4, 6, 7, effektivt skaper et vindu med forbedret sensitivitet for sykdom. Derfor studiet av tiltak før, under eller etter intens trening med mål om å redusere sin innvirkning på NK cellefunksjonen er av spesiell interesse for velvære for idrettsutøvere post-konkurranser. 8, ved omtrent 1% av de hvite blodlegemer rommet; 2) NK-celler er svært følsomme for stress og stole på konstant utsettes for fysiologiske betingelser for å være levedyktig, stabil under eksperimentering; og 3) standard analyser for å fastslå NK celle cytotoksisitet, for eksempel Ficoll gradienter 9 og slipp analyser 10, er upålitelige og inkonsekvent. Den iboende variabilitet av humane prøver bare sammensatte disse problemene. Ferske humane prøver samlet inn i løpet av intervensjoner er ganske regulert og vanskelig å skaffe, i hvert fall i forhold til dyr prøver eller udødeliggjorte cellelinjer. Dette reduserer mulighetene til å gjenta eksperimenter eller legge til deltakere i studiekohorten å nå betydelige statistiske terskler. Sammen er disse problemene støtter behovet for en strømlinjeformet protokoll som tillater for både høy gjennomstrømming og høy pålitelighet analyse av NK celle lytisk aktivitet i humane prøver. Vi etablerte en arbeidsflyt som forkorter tiden som er nødvendig for å identifisere, isolere og test NK-celler fra humant fullblod mens eksponering for utenforliggende faktorer minimeres. Fremgangsmåten optimaliserer bruken av to instrumenter, en magnetisk basert cellesorterer og en avbildnings strømningscytometer, og en analysespesifikk, optimalisert T: E ratio for å tillate påvisning av reduksjon eller økning av NK-celle cytotoksisitet.

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			<td height="21" style="height:21px;width:371px;">K-562 lymphoblasts</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">CCL-243</td>
			<td style="width:585px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Media</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">30-2005</td>
			<td>High glucose, with L-Glutamine, with HEPES, Sterile-filtered</td>
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			<td height="42" style="height:42px;">Alpha Minimum Essential medium</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:136px;">CRL-2407</td>
			<td style="width:585px;">Without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Trypan Blue Solution 0.4%</td>
			<td style="width:183px;">Amresco</td>
			<td style="width:136px;">K940-100ML</td>
			<td>Tissue culture grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Propridium Iodide Staining Solution</td>
			<td style="width:183px;">BD Pharmingen</td>
			<td style="width:136px;">51-66211E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Vybranto DiO cell-labeling solution</td>
			<td style="width:183px;">Vybranto</td>
			<td style="width:136px;">V-22886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Pro Separator</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-092-545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Running Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Washing Buffer</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-092-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">autoMACS Columns</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Blood CD56 MicroBeads, human</td>
			<td style="width:183px;">Miltenyi Biotec</td>
			<td style="width:136px;">130-090-875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageStream X Mark II Imaging Flow Cytometer</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
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			<td height="21" style="height:21px;">Speedbeads</td>
			<td style="width:183px;">Amnis Corporation</td>
			<td style="width:136px;">400030</td>
			<td></td>
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			<td height="21" style="height:21px;">0.4-0.7% Hypochlorite (Sterilizer)</td>
			<td style="width:183px;">VWR</td>
			<td>JT9416-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coulter Clenz</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td>8546929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropanol (Debubbler)</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;">1.3704</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INSPIRE Software</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td>Version Mark II, September 2013</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ideas Application Software</td>
			<td style="width:183px;">EMD Millipore</td>
			<td style="width:136px;"></td>
			<td>Version 6.1, July 2014</td>
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flow cytometric analysis of natural killer cell lytic activity in human whole blood

NK-celle cytotoksisitet er en mye brukt tiltak for å bestemme virkningen av utvendig intervensjon på NK-cellefunksjonen. Imidlertid kan den nøyaktighet og reproduserbarhet av denne analysen overveies ustabil, enten på grunn av bruksfeil eller på grunn av følsomheten av NK-celler til eksperimentell manipulasjon. For å eliminere disse problemene, ble en arbeidsflyt som reduserer dem til et minimum etablert og er presentert her. For å illustrere, fikk vi blodprøver, på ulike tidspunkter, fra løpere (n = 4) som ble sendt til en intens anfall av trening. Først blir NK-celler samtidig er identifisert og isolert gjennom CD56 merking og magnetbaserte sortering, direkte fra fullblod og fra så lite som en milliliter. De sorterte NK-celler blir fjernet fra eventuelle reagens eller lokking av antistoffer. De kan telles for å etablere en nøyaktig NK-celletall per milliliter blod. For det andre kan de sorterte NK-celler (Effektor-celler eller E) blandes med 3,3&#39;-Diotadecyloxacarbocyanine perklorat (DIO) merket K562 celler (målcellene eller T) i en analyse-optimal 1: 5 T: E ratio, og analysert ved hjelp av en bildestrøm-cytometer som gjør det mulig for visualisering av hver hendelse og eliminering av falske positive eller falske negativer (for eksempel dubletter eller effektorceller). Denne arbeidsflyten kan være ferdig i ca 4 timer, og gir svært stabile resultater selv når du arbeider med humane prøver. Når tilgjengelig, kan forskergrupper teste flere eksperimentelle inngrep i mennesker, og sammenligne målinger over flere tidspunkter uten å ødelegge data integritet.

Fastsettelse av NK-celletall Effekten av tunge som kjører på NK-celletall i fullblod ble målt ved hjelp av utøvelse protokoll beskrevet i figur 2. Blodprøver ble tatt før trening, umiddelbart etter trening, 1,5 timer etter trening, og til slutt 24 og 48 timer etter den første blodprøvetaking. Konsentrasjonen av NK-celler per milliliter av fullblod ble målt for hver løper (figur 3A) og i snitt (figur 3B) for hvert tidspun...

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.

Flowcytometrisk analyse av Natural Killer Cell Lytisk aktivitet i humant fullblod

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-oppløsning Imaging of Natural Killer Cell Immunologiske Synapse på en Glass-støttet Planar lipidbilag

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 ...

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

Hel-dyr Imaging og strømningscytometriske Teknikker for analyse av Antigen-spesifikke CD8 + T celle responser etter Nanopartikkel Vaksinasjon

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

Isolasjon og flowcytometrisk analyse av immunceller fra iskemisk Mouse Brain

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

Isolering og flowcytometrisk analyse av gliom-infiltrerende perifere mononukleære blodceller

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

En enkel Flowcytometriske metode for å måle glukoseopptak og Glukose Transporter Expression for Monocyttnivåene subpopulasjoner i fullblod

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

Flowcytometrisk analyse av partikkelbundet Bet v 1 Allergen i PM10

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

Isolasjon og Flowcytometriske analyse av endocervikale Gamma Delta T-celler

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

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

Én celle analyse av Immunophenotype og cytokin produksjon i eksterne fullblod via masse cytometri

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

Karakteristikk av menneskelig Monocyte delsett av hele blod flyte cytometri analyse

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

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Måle fagocytose av Aspergillus fumigatus Conidia av menneskelige leukocytter bruker Flow flowcytometri

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...