Name: a simple flow cytometric method to measure glucose uptake and glucose transporter expression for monocyte subpopulations in whole blood
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この研究は、サポートされている賞番号AI027757の下エイズ研究のためのワシントン大学のセンター（CFAR）、NIH資 ​​金提供プログラムからHIVや肝炎ウイルス学研究（ACH 2）オーストラリアセンター、2010発達グラント（CNIHR）によって賄われていました以下のNIH研究所およびセンター（NIAID、NCI、NIMH、NIDA、NICHD、NHLBI、NIA）によります。 CSPはCNIHRとACH 2助成金の受取人です。 SMCは、オーストラリア国立保健医療研究評議会（NHMRC）主な研究フェローシップの受領者です。作者は感謝しバーネット研究所によって受信されたビクトリア朝の運用インフラ支援プログラムのこの作業への貢献を認めます。私たちは訓練と技術的なアドバイス、フローサイトメトリーのためのAMREPフローサイトメトリーコアファシリティから下座PaukovicとエヴァOrlowski - オリバーの支援を認めます。私たちは、メディアのコーチングやビデオ撮影の組織のためのアンガス・モーガンに感謝します。私たちの感謝の気持ちジェシーマッソンとジハード・アブドゥルアジーズK. Alzahraniにビデオ撮影時のラボの支援のために。我々は重要な方法論的なアドバイスを提供した医学研究科で博士デビッドSimar、UNSW、オーストラリアの努力に感謝します。 CSPは感謝したいと思い<a href="http://www.nice-consultants.com/">www.nice-consultants.comを</a>グラフィック協議するため。 レビューアの貢献： CSPは、設計と実験を行い、分析し、データを解釈し、プロジェクトを考案し、原稿を書きました。 JJAは、データを解釈し、原稿を書きました。 TRBは、原稿を書きました。 JMMは、データを解釈し、重要な知的な提案を行い、原稿をレビューしました。 SMCは、データを解釈し、重要な知的提案を行い、原稿をレビューしました。

注：HIV感染およびHIV非感染被験者は、メルボルンのアルフレッド病院で感染症のユニットから募集した、VIC、オーストラリア、そして地域社会から、それぞれ。インフォームドコンセントは、全ての参加者から入手した、と研究はアルフレッド病院研究倫理委員会によって承認されました。 単球及び単球亜集団1. GLUT1細胞表面検出<ol><li>クエン酸ACD-B抗凝固剤チューブに...

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ここで説明するプロトコルは、全血における単球及び単球亜集団によるグルコーストランスポーターの発現と蛍光グルコースアナログの取り込みを調べるための簡単​​な方法を詳しく説明します。全血中の2-NBDG取り込みを評価することにより、この技術は、 インビボでのものと同様の条件を可能にします。以前の研究では、密度遠心分離17によって、全血から分離した単球?...

単球は、急速に感染し、炎症1の部位に動員されている人間の自然免疫系の主要な構成要素です。単球の活性化は、病原体による急性の損傷を制限するために重要であり、また、アテローム性動脈硬化症2、癌3、およびHIV 4,5を含むいくつかの慢性疾患の病因の中心です。 休止および活性化単球の代謝は休止単球は、解糖代謝（乳酸へのグルコースの、すなわち 、発酵）を利用した酸化的代謝および活性化単球を利用することで、劇的に異なる6。単球の活性化は、解糖系代謝7増加したグルコース取り込みを可能にするグルコース輸送体の発現を誘導します。単球のグルコーストランスポーター1（GLUT1）は、活性化の間にアップレギュレートさそのような輸送体であり、その発現は、Vにおける前炎症性サイトカインの産生をもたらすことが示されていますITROと肥満マウス8の脂肪組織インチカポジ肉腫関連ヘルペスウイルスによって単球細胞株の感染はGLUT1 9の細胞のアップレギュレーションにつながる、と私たちは最近、慢性HIV感染の間にGLUT1を発現する単球の増加割合が未処理と併用抗レトロウイルス療法で処置した感染の10の間に存在していることを示しました。まとめると、これらの研究は、単球によるグルコース取り込み及び解糖代謝は、多くの炎症性疾患の重要な側面であることを示しています。従って、簡単な方法は、恒常性中単球GLUT1発現およびグルコース取り込みを測定し、炎症性疾患は、研究者の幅広い使用の可能性が高いです。 ヒト単球は、細胞表面マーカーCD14及びCD16 11,12の発現差によって調べることができる3つの別個のサブセットで構成され、不均一です。クラシック単球は、CD14の高いレベルを発現するが、CDを発現しません16（CD14 ++ CD16 - ）、中間の単球は、CD14の高レベルおよびCD16（CD14 ++ CD16 +）の中間レベルを発現し、非古典的単球は、CD14の低レベルとCD16の高レベル（CD14を発現し+ CD16 ++）。 CD16を発現する単球は、CD16に比べCD16 +単球を、と呼ばれる-単球は、高い炎症性サイトカインの発現とをより効果的に存在する抗原13,14能力を持っています。単球の約10％は、炎症15の間に観察されたより高い割合との恒常性の間にCD16を発現します。単球の亜集団は、特定の疾患状態と関連していると、病気や疾患の進行16の有用な生物学的マーカーである可能性があります。 私たちの目標は、PHYに近い条件でのヒト単球と単球亜集団によるグルコーストランスポーターの発現とグルコースの取り込みを測定することができる方法を同定することでしたできるだけsiological条件。これらの方法は、生理学的条件19に比べてタンパク質の発現が変化していることができ、単離された単球を検討しても以前の研究では、単球のグルコース輸送体の発現とグルコースの取り込み17,18を測定し、以前の研究では、ヒト単球亜集団を検討していません。フローサイトメトリーのマルチパラメトリックフローを使用して、我々は全体の未操作の血の中に（CD14およびCD16の発現に基づく）総単球及び単球亜集団によるグルコーストランスポーターの発現と蛍光グルコースアナログ2-NBDGの取り込みを調べるための方法を説明します。

<table border="0" cellpadding="0" cellspacing="0" width="570">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:140px;">VACUETT Tube 9 ml ACD-B anticoagulant tubes</td>
			<td style="width:147px;">Greiner Bio-One GmbH</td>
			<td style="width:139px;">455094</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:140px;">5 ml sterile polypropylene tubes</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">352063</td>
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			<td height="41" style="height:41px;width:140px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:139px;">A7906</td>
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			<td height="41" style="height:41px;width:140px;">16% formaldehyde solution</td>
			<td style="width:147px;">Electron Microscopy Science</td>
			<td style="width:139px;">15710</td>
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			<td height="121" style="height:121px;width:140px;">BD FACS lysing solution (10X)</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">349202</td>
			<td style="width:147px;">Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4&#176;C)</td>
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			<td height="22" style="height:22px;width:140px;">anti-CD3-PE</td>
			<td style="width:147px;">BD Biosciences</td>
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			<td style="width:147px;">BD Biosciences</td>
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			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">FAB1418F</td>
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			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">IC0041F</td>
			<td style="width:147px;"></td>
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		<tr height="241">
			<td height="241" style="height:241px;width:140px;">2-NBDG</td>
			<td style="width:147px;">Life technologies</td>
			<td style="width:139px;">N13195</td>
			<td style="width:147px;">Suspend 5 mg of 2-NBDG into 1 ml of deionized water to make a 14.60 mM stock solution (keep for up to 6 months at 4&#176;C). To make the working 2-NBDG concentration, dilute stock 1/100 with 1X DPBS. Cover with foil. (store for up to 1 week at 4&#176;C)</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:140px;">Dulbecco&#8217;s Phosphate Buffered Saline (1X)</td>
			<td style="width:147px;">Life technologies</td>
			<td style="width:139px;">14190-144</td>
			<td style="width:147px;">To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4&#176;C)</td>
		</tr>
	</tbody>
</table>

a simple flow cytometric method to measure glucose uptake and glucose transporter expression for monocyte subpopulations in whole blood

単球は、特定の慢性炎症性疾患に関連する病原体および炎症によって活性化することができる自然免疫細胞です。単球の活性化は、エフェクター機能と増加したグルコース輸送体発現によって達成される解糖代謝への酸化から付随シフトを誘導します。この増加した解糖代謝はまた、訓練を受けた単球の免疫、先天性免疫記憶の形態で観察されます。単球によるグルコーストランスポーターの発現とグルコース取り込みを調べるインビトロプロトコルが記載されているが、いずれも全血マルチパラメトリックフローサイトメトリーによって検討されていません。 （中間体（CD14 ++ CD16 +）および非古典-私たちは、総単球および古典（CD14 ++ CD16）によって全血における蛍光グルコースアナログ2-NBDGの取り込みを測定するためのマルチパラメトリックフローサイトメトリープロトコルを記述しますCD14 + CD16 ++）、単球亜集団。この方法は、恒常性および炎症性疾患の間の総単球及び単球亜集団のためのグルコーストランスポーターの発現及びグルコース取り込みを調べるために使用することができ、容易に血液中の他の白血球および白血球亜集団のためのグルコース取り込みを調べるために改変することができます。

補償は、蛍光波及を防ぐために、個々の蛍光色素のために実行する必要があります。単球は、最初の前方および側方散乱に基づいてゲートすることによって濃縮されます。以前に10を報告したように提示したプロットは、6人以上の参加者からの全血で行われ、少なくとも6つの独立した実験の代表で、図1Aは、CD3以内ゲーティングによるT細胞の細?...

Watch this Scientific Journal Video about A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood at JoVE.com

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

The overall goal of this methodology is to measure the uptake of glucose and its transporter on different monocyte subpopulations. This method can be used to answer key questions in the immunology field, such as What is the importance of immune cell metabolism in disease outcomes? The main advantages of these techniques are it is quick, easy to perform, requires no reactive material, and requires only a small volume of blood.Within one hour of collecting the blood samples, in citrate ACD-B anticoagulant tubes, place the samples into a biological safety cabinet. Next, add 100 microliters of blood from each sample into individual polypropylene tubes, and treat the cells with two milliliters of 1X lysing solution on ice, pipetting gently to mix. After 15 minutes, centrifuge the cells and follow this with two more spins in wash buffer.After the second wash, use a pipette to carefully remove as much of the supernatants as possible, and re-suspend the pellets in 100 microliters of wash buffer on ice. To identify the specific monocyte subpopulations, stain the cells with five microliters of the appropriate antibodies per 100 microliters of cells, for 30 minutes on ice in the the dark. At the end of the incubation, wash the samples two times in wash solution and fix the cells with 200 microliters of 0.5%formaldehyde in PBS.Then, analyze the cells within 24 hours on a flow cytometer capable of detecting four colors. To assess the glucose uptake of the monocyte subpopulations, transfer 90 microliters of blood from each sample into individual polypropylene tubes, and add 10 microliters of the fluorescent glucose analog of interest to the blood. Flick the tubes gently to mix, and cover the samples with aluminum foil.Then, place the cells at 37 degrees Celsius for 15 to 30 minutes, followed by their immediate transfer to ice. Next, add four milliliters of FACS lysing solution to the tubes, and centrifuge the cells. Then, perform a second centrifugation in wash buffer.Decant the supernatant, and stain the cells with the appropriate antibodies of interest for 30 minutes at four degrees Celsius in the dark. Then, wash the samples in four milliliters of ice-cold wash buffer, re-suspend the pellets in 200 to 300 microliters of ice-cold PBS, and analyze the tubes within 10 minutes. For flow cytometric analysis, first use the unstained and individually stained control samples to set the compensation.Next, in the appropriate single-cell analysis software, open the first sample in a forward by side scatter plot, and gate the monocytes as illustrated. Then, double-click the gated monocyte population, and gate the CD3-negative population. Now double-click on the CD3-negative population, and set the x-axis to display the CD14 staining, and the y-axis to display the CD16 staining.Next, to measure the uptake of glucose or its transporter in a specific monocyte subpopulation, generate the appropriate histograms. Then, where defined cell populations exist, use the control FITC igG samples to set the gate and determine the total percentage of CD14-positive monocytes in each sample. Here, the initial gating strategy for isolating the monocytes by cell scatter and T-cell exclusion by gating within the CD3-negative population, is shown.The monocytes are then gated for CD14 expression alone, or in combination with CD16 to identify the total monocytes or monocyte subpopulations. Distinct populations of CD14-positive Glut-1 glucose transporter positive monocytes may be observed, most notably in the circulating white blood cells obtained from HIV-positive individuals. CD14-positive Glut-1 positive cells may also be observed within specific monocyte subsets in HIV-uninfected individuals, although these populations are more pronounced in HIV-positive patients.As expected from the glucose transporter expression data, the glucose analog 2-NBDG, is also taken up in higher quantities by cells from HIV-positive individuals. Once mastered, this technique can be completed within one-and-a-half hours if performed properly. While attempting this procedure, it is important to limit light exposure to all regions.Following glucose analog uptakes, cells can be stained with cell surface antibodies to identify specific immune populations that you're interested in. After its development, this method can be used to determine how HIV affects immune cell metabolism, can also be explored by other researchers, in different fields, such as immunology, virology, and cancer research. After watching this video, you should have a good understanding on how to detect glucose transporter one, and glucose uptake in immune cells.Don't forget that when working with infectious agents, standard safety procedures should be employed.

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