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.

<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. 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. W., Cieslak, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+natural+killer+cell+subsets+and+acute+exercise:+a+brief+review.">Human natural killer cell subsets and acute exercise: a brief review.</a> Exerc Immunol Rev. 14, 8-23 (2008).</li><li>Westermann, J., Pabst, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+lymphocyte+subsets+and+natural+killer+cells+in+the+human+body.">Distribution of lymphocyte subsets and natural killer cells in the human body.</a> Clin Investig. 70 (7), 539-544 (1992).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+leucocytes+from+human+blood.+Further+observations.+Methylcellulose,+dextran,+and+ficoll+as+erythrocyteaggregating+agents.">Isolation of leucocytes from human blood. 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. 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=Effect+of+blueberry+ingestion+on+natural+killer+cell+counts,+oxidative+stress,+and+inflammation+prior+to+and+after+2.5+h+of+running.">Effect of blueberry ingestion on natural killer cell counts, oxidative stress, and inflammation prior to and after 2.5 h of running.</a> Appl Physiol Nutr Metab. 36 (6), 976-984 (2011).</li><li>Millard, A. L., 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=Brief+Exercise+Increases+Peripheral+Blood+NK+Cell+Counts+without+Immediate+Functional+Changes,+but+Impairs+their+Responses+to+ex+vivo+Stimulation.">Brief Exercise Increases Peripheral Blood NK Cell Counts without Immediate Functional Changes, but Impairs their Responses to ex vivo Stimulation.</a> Front Immunol. 4, 125 (2013).</li><li>Janeway, C. 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>

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 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 ...

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

Research

JoVE Journal

Immunology and Infection

12.3K 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

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

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 been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

グラスサポート平面脂質二重層上のナチュラルキラー細胞免疫シナプスの超解像イメージング

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 ...

免疫・感染学

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

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

ナノ粒子ワクチン接種後の抗原特異的CD8 + T細胞応答の分析のための全動物イメージングとフローサイトメトリー技術

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

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

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

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

神経膠腫浸潤末梢血単核細胞の単離およびフローサイトメトリー分析

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

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

シンプルなフローサイトメトリー法は、全血における単球亜集団のためのグルコース取り込みおよびグルコーストランスポーターの発現を測定するために

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

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 diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10における粒子結合のBet V 1アレルゲンのフローサイトメトリー分析

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

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

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

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血によって人間の Monocyte のサブセットの解析フローのフローサイトメトリー解析

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

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

好中球系統細胞の質量細胞法解析に対する全骨髄の調製

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

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

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.