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.

참고 : 모든 혈액 수집 절차는 애팔 래 치아 주립 대학 (ASU) 임상 시험 심사위원회 (IRB)에 의해 규정 된 지침에 따라 실시 하였다. 1. 전체 혈액 컬렉션 <ol><li> 공인 phlebotomist는 세계 보건기구 (WHO)의 가이드 라인에 따라 혈액을 채취 있습니다. </li><li> 디 에틸렌 디아민 산 칼륨을 함유 한 4 mL의 혈관 (K 2 EDTA)으로 혈액을 채취. 제조업체의 지시에 따라 혈관을 전환. 분리 ...

<ol>
	<li>Cerwenka, A., Lanier, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cells,+viruses+and+cancer.">Natural killer cells, viruses and cancer.</a> Nat Rev Immunol. 1 (1), 41-49 (2001).</li><li>Nieman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise,+infection,+and+immunity.">Exercise, infection, and immunity.</a> Int J Sports Med. 15, 131-141 (1994).</li><li>Romeo, J., Warnberg, J., Pozo, T., Marcos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity,+immunity+and+infection.">Physical activity, immunity and infection.</a> Proc Nutr Soc. 69 (3), 390-399 (2010).</li><li>Nieman, D. 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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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A., Travers, P., Walport, M., Shlomchik, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunobiology: The Immune System in Health and Disease. 5th edn. , (2001).</li></ol>

본 연구에 기재된 방법은 직접 (특히이 경우, 장시간 운동) 자극에 응답하여 개인의 NK 세포의 특이 적 활성을 측정한다. 일반적으로, NK 세포는 마커의 조합을 사용하여 분류 밀도 구배 또는 셀을 사용하여 하나의 혈액으로부터 분리된다. 그들은 시간이 걸리는 다수의 조작을 수반 한 사용자 심하게 의존하는 이러한 방법이 널리 사용되지만, 그들은 여러 단점을 가지고있다. 그 결과, 과도한 스트?...

자연 살해 세포는 선천성 면역계의 필수 요소이다. 그들은 매우 조절 동안, 인식하고 세포 간 접촉을 통해 사전 활성화 일없이 비정상 세포를 제거하는 기능을 갖는다. 따라서, 그들은 감염에 대한 방어의 빠른 라인을 형성한다. 특히 심한 운동은, 일시적으로 면역 시스템 2, 3, 4, 5를 누르는 것으로 나타났다. NK 세포가 효과적으로 질병 향상된 감도의 창을 생성이 효과 4, 6, 7, 특히 쉽다. 따라서, 개입의 연구는 전, 중 또는 NK 세포 기능에 미치는 영향을 줄이는 목적으로 강렬한 운동 후하는 운동 선수 후 대회의 복지에 대한 특별한 관심입니다. 8 드물다 1); 2) NK 세포 스트레스에 매우 민감하고 실험 기간 동안 생존하고 안정적으로 유지하는 생리 학적 조건에 노출에 의존하는 상수; 3) 표준 분석은 피콜 구배 9로, NK 세포의 세포 독성을 확인하고 분석 (10)를 해제, 신뢰할 수와 일치하지 않습니다. 인간 샘플의 고유 한 변화는 이들 이슈 인 화합물. 중재 동안에 수집 된 신선한 인간 샘플 적어도 동물 샘플 또는 불멸화 세포주에 비해, 수배 상당히 규제하고 어렵다. 이 실험을 반복하거나 통계적으로 유의 임계 값에 도달하기 위해 연구 코호트에 참가자를 추가 할 수있는 기회를 감소시킨다. 종합적으로, 이러한 문제는 fo를 간소화 할 수있는 프로토콜의 필요성을 지원R 모두 높은 처리량과 인간 시료에서 NK 세포 용균 활성의 높은 신뢰도 분석. 우리는 식별 외부 인자에 대한 노출을 최소화하면서 인간의 전혈로부터 분리하고 시험 NK 세포에 필요한 시간을 단축 워크 플로우를 구축. 감소 또는 NK 세포의 세포 독성의 증가를 검출 할 수 있도록 E 비율 : 상기 방법은 두 기기, 자기 기반의 셀 소터와 사이토 촬상 흐름 및 분석 특정 최적화 T의 사용을 최적화한다.

<table border="0" cellpadding="0" cellspacing="0" width="1275">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Speedbeads</td>
			<td style="width:183px;">Amnis Corporation</td>
			<td style="width:136px;">400030</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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

NK 세포는 NK 세포 독성 세포 기능에 대한 외부 간섭의 영향을 결정하기 위해 널리 사용되는 척도이다. 그러나,이 분석의 정확성 및 재현성 때문에 사용자의 오류로 인해 또는 실험 조작에 NK 세포의 감도 역시 불안정한 것으로 간주 될 수있다. 이러한 문제를 제거하기 위해 최소로 줄여 워크 플로우에 설립되었으며 여기에 표시됩니다. 우리는 선수에서, 다양한 시점에서 혈액 샘플을 얻어 설명하기 위해 (N = 4) 운동의 강렬한 한판 승부에 제출 된 그. 첫째, NK 세포를 동시에 확인하고 직접 전체 혈액에서와 같은 작은 하나 밀리리터에서, CD56 태그 및 자기 기반의 정렬을 통해입니다. 정렬 된 NK 세포는 시약 또는 상한 항체의 제거됩니다. 이들은 혈액 밀리리터 정확한 NK 세포 수를 설정하기 위해 계산 될 수있다. 둘째, 분류 NK 세포 (이펙터 세포 또는 E)를 3,3&#39;- Diotade와 혼합 될 수있다각 이벤트의 시각화 및 위양성의 제거를 허용하는 촬상 유동 세포 계측기를 사용하여 E 비율 분석 : 5 T : cyloxacarbocyanine 과염소산 (DIO)은 분석 최적 1의 K562 세포 (표적 세포 또는 T)를 태그 또는 (예 : 이중선 또는 이펙터 세포와 같은) 위음성. 이 워크 플로는 약 4 시간에 완료, 심지어 인간의 샘플 작업을 매우 안정적인 결과를 허용 할 수 있습니다. 가능한 경우, 연구 팀은 인체에서 여러 가지 실험적인 개입을 테스트, 데이터의 무결성을 손상시키지 않고 여러 시점에 걸쳐 측정을 비교할 수 있습니다.

NK 세포 수의 결정 전혈에서 NK 세포 수 무거운 실행의 효과는도 2에 기재된 운동 프로토콜을 사용하여 측정 하였다. 혈액 샘플은 초기 채혈 후 최종적으로 24, 48 시간을 1.5 시간 운동 후, 즉시 운동 후, 운동 전에 그려진 하였다. 전혈 밀리리터 NK 세포의 농도는 각각의 러너 (도 3A)에 대한 각 타임 포인트에 대한 평균 (도 3b)상에서 ?...

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.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

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

유리 - 지원 평면 지질 이중층에 자연 살해 세포 면역 시냅스의 슈퍼 해상도 이미징

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 ...

연구

면역학 및 감염병학

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

나노 입자 백신 접종 후 분석 항원 특이 CD8 + T 세포 반응에 대한 전체 동물 이미징 및 유세포 기술

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

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

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

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

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

간단한 유세포 방법은 전체 혈액에서 단핵구 부분 집단에 대한 당 흡수 및 포도당 운송업자 발현을 측정하는

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

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

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

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

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

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

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

Immunophenotype 및 Cytokine 생산 대량 Cytometry 통해 주변 전 혈의 단일 세포 분석

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

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

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