这项研究是由澳大利亚中心艾滋病毒和肝炎病毒研究从华盛顿艾滋病研究中心大学（CFAR），根据奖号AI027757美国国立卫生研究院资助的程序，它支持（ACH 2）和2010年的发展拨款（CNIHR）资助通过下面的NIH研究所和研究中心（NIAID，NCI，镍氢电池，NIDA，NICHD，NHLBI，NIA）。 CSP是CNIHR和ACH 的赠款获得者。 SMC是澳大利亚（NHMRC）主要研究奖学金的国家健康和医学研究理事会的收件人。作者非常感谢由伯纳特机构收到了维多利亚经营性基础设施项目的支持这项工作的贡献。我们承认格扎Paukovic和伊娃ORLOWSKI - 奥利弗从AMREP流式细胞仪核心设施流式细胞仪培训和技术咨询协助。我们感谢安格斯摩根媒体教练和视频拍摄的组织。我们的感激杰西马森和讨伐异教徒阿卜杜勒阿齐兹K. Alzahrani在视频拍摄过程中的实验室协助。我们感谢大卫博士SIMAR在医学科学，新南威尔士大学，澳大利亚谁提供关键的方法建议，学校的努力。 CSP想感谢<a href="http://www.nice-consultants.com/">www.nice-consultants.com</a>用于图形磋商。 作者的贡献: CSP构思项目，设计并进行了实验，分析和解释数据，并且写了稿子。 JJA解释数据和撰写文章。 TRB写的稿子。 JMM解释的数据，提出关键知识的建议，并审查了原稿。 SMC解释的数据，做出关键的智力建议并审查了原稿。

注:艾滋病病毒感染者和艾滋病病毒感染者是从传染病股在阿尔弗雷德医院在墨尔本，澳大利亚招募，并从当地社区，分别为。从所有参与者获得知情同意书，研究批准了阿尔弗雷德医院研究伦理委员会。 在单核细胞和单核细胞亚群1.细胞的Glut1表面检测<ol><li>收集血液柠檬酸ACD-B的抗凝管并开始实验在生物安全柜收集1小时之内。 </li><li>加入100微升血液聚丙烯管。加2ml 1X的裂解液（见材料表 ）来管，而冰，移液轻轻混合。孵育在冰上15分钟。离心在220×g离心5分钟。 </li><li>倒出并通过加入大约2-4毫升洗涤溶液（在1×PBS中的0.5％BSA）并在220 xg离心离心5分钟洗涤两次。 </li><li>使用管道TTE要小心地取出尽可能多的洗涤液成为可能。在100微升洗涤液管放置在冰上重新挂起。 </li><li>确定具体的单核细胞亚群染色细胞与步骤1.4制得每100抗体微升细胞悬浮液的以下量:5微升抗CD3-PE，5微升抗-CD14-APC，5微升抗CD16-PECy7，5微升的Glut1 -FITC或的IgG2b-FITC（同型对照管）。 </li><li>在冰上放置在黑暗中30分钟。用洗涤液洗2次。 200-300微升在1X PBS在0.5％甲醛固定。 </li><li>以下激发和发射波长在24小时之内流式细胞仪能够检测4种颜色流分析:FITC（488，530），PE（488，575），PECy7（488，780），APC（633，660），10。 </li></ol> 2.葡萄糖摄取单核细胞<ol><li>收集在步骤1.1中的聚丙烯管移液器90微升的血液。添加10微升14.60μM2 NBDG工作液来90微升的血液（1.46毫米终浓度），并轻轻一抖混合。这是非常关键的覆盖管用铝箔限制2 NBDG曝光。 </li><li>孵育在37℃下在黑暗中15-30分钟，然后立即置于冰上。加4毫升1×FACS裂解解决管在冰冷。离心在4 220 XG °C为5分钟。 </li><li>通过加入4毫升洗涤溶液（0.5％在1×PBS中的BSA）洗涤一次。离心在4 220 XG °C为5分钟。倒出并在冰上的地方。 </li><li>染色细胞用抗体:5微升抗CD3-PE，5微升抗-CD14-APC和5μl抗CD16-PECy7。混合，并在冰上放置在黑暗中30分钟。 注:在此期间确保流式细胞仪已经准备好进行实时分析。获得以下几方面激发和发射波长范围内的单元格:2 NBDG（488，530），PE（488，575），PECy7（488，780），APC（633，660）。 </li><li> 4ml冰冷的洗涤缓冲液（在1×PBS中的0.5％BSA）添加到管中。离心220 XG洗一次在4 °C为5分钟。倒出并加入200-300微升冰旧的PBS，并保持在黑暗冰（用铝箔覆盖）。在流式细胞仪采用激发和发射波长设定在2.4步10分钟的流量进行分析。 </li></ol> 3.数据采集与分析注意:假设流式细胞仪和数据分析方面的知识。 <ol><li>用流式细胞仪能够至少4色分析的流，使用未染色和单独地染色样品设置的补偿。 注意:使用FITC标记的CD4和CD14单染色可用于的Glut1和2- NBDG补偿。 </li><li>建立和采集取样前标注相应的窗口。绘制围绕单核细胞群体的栅极，并获得每个样品100,000-300,000事件在中等速率。每个样品赔偿50,000个事件就足够了。 注:补偿之前，可以进行采样采集或在单细胞分析软件，以下的标准程序。 </li><li>出口和数据保存到一个适当的位置。打开的单细胞分析软件如的FlowJo或其他分析软件（ 参考图1）和拖放样品作为指定（ 参考图2）。 </li><li>双击打开文件（ 参考图3）。画一个圆门了一种基于单核细胞和图1A和补充图4所示侧向散射特性。双击单核细胞的人口。观察周围画了一个CD3箱-人口（ 参考图4）。 </li><li>双击CD3 -人口。观察单核细胞亚群上的"X"轴选择CD14-APC和CD16-PECy7上的"Y&#39;-轴，并相应标签（ 参考图5）。 </li><li>那里有没有明显阳性和阴性群，测量的Glut1在特定的单核细胞亚群表达或2-NBDG摄取。通过减去同种型和不2- NBDG背景（ 参考图6）确定的Glut1和2- NBDG的平均荧光强度（MFI）。 </li><li>其中，定义种群存在，则使用的IgG2b-FITC来设置的栅极，并确定百分比阳性细胞（ 图3）。 注:使用此方法来分析总CD14 +单核细胞。由于2-NBDG摄取通常是由在荧光移位标记强度数据最好由MFI和直方图表示。 </li></ol>

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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=Emerging+Role+and+Characterization+of+Immunometabolism:+Relevance+to+HIV+Pathogenesis,+Serious+Non-AIDS+Events,+and+a+Cure.">Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure.</a> J Immunol. 196 (11), 4437-4444 (2016).</li></ol>

The authors have nothing to disclose.

这里描述的协议细节的简单方法来检查在全血中的单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和荧光葡萄糖类似物的摄取。通过在全血评估-2- NBDG摄取，此技术允许类似于在体内条件。先前的研究中通过密度离心17日全血中分离的单核细胞检测6 NBDG摄取。然而，这项研究并没有审查的单核细胞亚群，并从全血中单核细胞的分离可以潜在地改变某些细胞表面分子19的表达。放射性葡萄糖示踪剂也已用于测量的葡萄糖20,21单核细胞摄取，但是单核细胞必须为此方法和放射性的用途来预先分离需要显著安全预防措施。我们的协议采用常规的生物安全程序并且是最低限度地操纵，从而允许2-NBDG的流式细胞测量由单核细胞和单核细胞亚群在体内条件模仿吸收。 2-NBDG进入糖酵解途径和已经显示被细胞代谢成非荧光分子22。因此，重要的是通过保持在4℃下冷​​却的细胞，以限制后代谢-2- NBDG孵育。 6- NBDG是另一个荧光葡萄糖类似物，可使用，但因为它不进入糖酵解途径，因此，不能准确地反映了细胞23的生物能量状态是不太有用。 如果利用重叠光谱荧光，补偿变得至关重要，以防止荧光外溢。在这个协议中，我们使用与CD14和CD16逐个染色来设置补偿参数的单核细胞，但也可以使用补偿珠进行的细胞表面标记物的补偿。 粒细胞可表达CD16，但可以通过门控出被排除CD15表达细胞。然而，基于光散射性质可以限制粒的数量的单核细胞的严格选通包括在分析中。如果流式细胞仪是可用更多的通道，粒细胞标记物CD66b也可用于排除在分析粒细胞。 在这项研究中所用的的Glut1抗体结合至细胞表面的表位，因此，不结合于细胞内的Glut1。结合于细胞内的Glut1表位的抗体可被用于测量总的Glut1单核细胞的表达，但细胞必须染色10之前透。除了单核细胞，该技术可以用来检查在血液中发现的其它白细胞葡萄糖摄取和代谢。使用此处描述的方法，我们进行了广泛研究T细胞摄取2-NBDG的，并且还通过NK细胞24检测2-NBDG摄取。对于成功侦破表达的Glut1和2 NBDG摄取的当务之急是要限制红血细胞裂解液的痕迹洗涤细胞用根据本协议过量洗涤缓冲液，并且我们发现FITC或APC标记的Glut1抗体给予比PE或PerCP缀合物更好的信号。我们还没有研究这方面的原因。 因为细胞即使在低温下是代谢活性，这是至关重要的下面，在37 2-NBDG孵化 ℃，在4℃下该管被直接保持在冰上和离心进行。在缺乏强有力2 NBDG信号，检查是否使用了正确的集中，在适当的时候降低了房间，生物安全柜和覆盖管带箔曝光。优化可以通过设置一个时间过程-2- NBDG摄取实验5，15，20，30，60，和90分钟是必需的。通常情况下，最佳的时间应为10〜60分钟，这取决于细胞类型和它们的活化状态。 与2 NBDG摄取试验的主要限制是光SENSitivity与它正在由细胞所利用的事实一起。因此，它以限制的样本数，以确保最后的样品是在第一个30分钟内进行分析是很重要的。的生物限制在于，即使的Glut1表达非经典的单核细胞，和的Glut1表达对非经典的单核细胞的频率比传统的单核细胞显著越大，在两个亚群10之间-2- NBDG摄取不存在任何差异。这就提出了其他生产过剩，如GLUT3和GLUT4表达于单核细胞可能参与葡萄糖代谢在不同疾病的设置的可能性。它也可能是的Glut1的活性也可被翻译调控交。 我们的流式细胞葡萄糖摄取协议通过放射性​​同位素标记的主要优点是该技术与免疫分析相结合，以确定和研究中SMAL免疫细胞的特异性亚群的能力血升容积。除了在APC缀合的抗的Glut1可以应用于同时分析的Glut1细胞表面表达和2- NBDG摄取。由于2-NBDG摄取的Glut1表达的改变以前尚未证实，但这种可能性不能被排除。 增加的葡萄糖摄取和代谢的免疫细胞是活化的T细胞和单核细胞25-27的一个标志。这些细胞可以在响应中激活病原体感染28,29，及炎症信号的条件，例如自体免疫疾病，如狼疮30-32，以及肥胖和糖尿病8,33。还需要用于癌症细胞存活，生长和转移34增加的葡萄糖代谢。值得注意的是，在免疫细胞的代谢失调已成为HIV感染的标志，并与免疫激活24，炎症10，和感染CD4 + T细胞35-37相关联。因此，这种方法将是感兴趣的不同的观众，包括那些在炎症介导的疾病，癌症，感染性疾病，免疫学和immunometabolism 38的兴趣。

单核细胞是正在迅速动员到感染和炎症1的位点的人的先天免疫系统的重要组成部分。单核细胞的活化是限制由病原体产生急性损害的关键，也是中央对多种慢性疾病，包括动脉粥样硬化2，3癌症和HIV 4,5的发病机制。 静息和活化的单核细胞的代谢显着不同，以利用氧化代谢利用糖酵解代谢（ 即 ，葡萄糖发酵成乳酸）静息单核细胞和活化的单核6。单核细胞的活化诱导葡萄糖转运，可以增加葡萄糖摄取糖酵解代谢7的表达。单核细胞葡萄糖转运蛋白1（的Glut1）是激活期间上调一个这样的转运，其表达已显示导致生产v中促炎细胞因子的itro和在肥胖小鼠8的脂肪组织。由卡波西肉瘤相关疱疹病毒单核细胞系感染导致9的Glut1细胞上调，我们最近发现，慢性感染艾滋病毒期间的Glut1表达的单核细胞百分比增加是未经处理及联合抗逆转录病毒疗法治疗的感染10时在座。总而言之，这些研究表明，葡萄糖摄取和由单核细胞糖酵解代谢许多炎性疾病的重要方面。因此，一个简单的方法来测量稳态期间的单核细胞的Glut1表达和葡萄糖摄取和炎性疾病可能是使用了广泛的研究人员。 人单核细胞是异质的，正在由能够由细胞表面标记物CD14和CD16 11,12的差异表达进行检查三个不同的子集。经典单核细胞表达CD14的较高水平，但不表达CD16（CD14 + CD16 - ），中间的单核细胞表达CD14的高水平和CD16（CD14 + CD16 +）的中间电平，和非经典的单核细胞表达CD14的水平低和CD16高的水平（CD14 + CD16 +）。表达CD16的单核细胞被称为CD16 +单核细胞，这相比CD16 -单核细胞有炎性细胞因子的高表达和能力，更有效地呈递抗原13,14。单核细胞的大约10％与炎症15中观察到的更高的百分比的动态平衡期间表达CD16。单核细胞亚群与某些疾病状态相关联，并且可能是疾病和疾病进展16的有用的生物标志物。 我们的目标是确定可以测量通过在尽可能接近的phy条件人单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和葡萄糖摄取的方法siological条件越好。以前的研究测量的单核细胞葡萄糖转运蛋白的表达和葡萄糖摄取17,18，虽然这些检测方法可以比较生理条件19已经改变蛋白表达分离的单核细胞，并没有以前的研究调查了人单核细胞亚群。使用多参数流式细胞术，我们描述的方法来检查该荧光葡萄糖类似物2-NBDG按总单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和摄取（基于CD14和CD16的表达）全未经处理的血液内。

<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>
			<td style="width:147px;"></td>
		</tr>
		<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>
			<td style="width:147px;"></td>
		</tr>
		<tr height="41">
			<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>
			<td style="width:147px;"></td>
		</tr>
		<tr height="41">
			<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>
			<td style="width:147px;"></td>
		</tr>
		<tr height="121">
			<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>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-CD3-PE</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">555340</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti CD14-APC</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">555399</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-CD16-PECy7</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">557744</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-Glut1-FITC</td>
			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">FAB1418F</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">IgG2b-FITC</td>
			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">IC0041F</td>
			<td style="width:147px;"></td>
		</tr>
		<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

单核细胞是可通过与某些慢性炎性疾病相关的病原体和炎症被激活先天免疫细胞。单核细胞激活诱导效应职能和免受氧化糖酵解来代谢随之而来的转变，伴随增加葡萄糖转运蛋白表达。这增加了糖酵解代谢也为单核细胞经过培训免疫力，先天免疫记忆形式观察。虽然在体外单核细胞检查葡萄糖转运蛋白表达和葡萄糖摄取的协议进行了说明，没有已被多参数流式细胞仪在全血检测。我们描述了荧光葡萄糖类似物2-NBDG摄取全血中总单核细胞和经典（CD14 + CD16 - ）测定的多参数流式细胞协议，中间体（CD14 + CD16 +）和非经典（ CD14 + CD16 +）单核细胞亚群。该方法可用于检查稳态和炎症性疾病中总单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和葡萄糖摄取，并且可以容易地修改，以检查血液中的其它白细胞和白细胞亚群的葡萄糖摄取。

补偿必须单独荧光染料，以防止荧光溢出来执行。单核细胞首先通过选通基于前向和侧向散射富集。给出的图是从六个或更多个参与者对全血进行至少六个独立实验的代表如先前报道10 图1A示出了由CD3内选通由细胞散射和T细胞的排斥的单核细胞的最初选通-人口。然后单核细胞被选通为单独或与CD16以确定总单核细胞或单核细胞亚群的组合CD14的表达分别如图1B和图1C，。 -应表示大约100倍以上CD经典的单核细胞（CD14 + CD16）:单核细胞亚群的分析，如先前所描述12以下命名法，应施加14 MFI大于同种型对照和CD16的MFI应类似于同种型对照;中间的单核细胞（CD14 + CD16 +）应该表达较同型对照约100倍，更大的CD14 MFI和大约10倍更大的CD16 MFI较同型对照;非经典的单核细胞（CD14 + CD16 +）应具有类似的MFI为CD14和同种型对照和比同种型对照大约100倍更大的CD16的MFI。无CD14和CD16的表达细胞是不被认为单核细胞，不应该被包括在门控。然后可检查葡萄糖转运蛋白表达门控单核细胞或单核细胞亚群。如图2中所示，CD14 +的Glut1 +单核细胞的不同群体可以从HIV感染的个体，其中，感染的特征在于炎症的慢性状态得到的细胞观察到的，最显着的。类似的，但难得的CD14 + +的Glut1细胞可能OBS在HIV感染的人（ 图3A）的特定的单核细胞亚群内erved，但在HIV +个体（ 图3B）更显着。值得注意的是，在不存在明显的种群，它可适当表示的结果以平均值或中位数荧光强度，这考虑到在的Glut1细胞表面表达的累积增加。 葡萄糖摄取可以通过比较全血与2- NBDG或用于选通单核细胞或单核细胞亚群的车辆控制孵育来评估。我们以前表明，单核细胞的大约50％是15分钟温育10之后2-NBDG阳性。这种吸收量可以检测2 NBDG无达到饱和，当单核细胞2 NBDG摄取的差异可能不再存在。由HIV感染和艾滋病的人+单核细胞分析2 NBDG摄取由HIV +者，细胞显示较高的摄取哪些是与所述的Glut1表达数据（ 图4 - 5）的协议。总的来说，这些结果表明，这里所描述的测定法可用于潜在地研究在引发炎症状态，例如糖尿病，心血管疾病，以及病毒和细菌感染的生物环境中的单核细胞的代谢活动。 <img alt="图1" src="/files/ftp_upload/54255/54255fig1.jpg" /> 图1:选通用来分析代表艾滋病毒和艾滋病病毒阳性的血液样本总量的单核细胞和单核细胞亚群战略的全血样品通过流式细胞仪收集的1小时内分析了单核细胞表面表达的Glut1。 （A）细胞基于前向和侧向散射特性和CD3表达门控。 （ 二 ）审查总单核细胞，CD3 -细胞，然后门控为CD14的表达。 （C </st荣&gt;）为了检验单核细胞亚群（古典，C;中间，我和非经典，NC）为艾滋病毒感染者和艾滋病病毒感染初次接受治疗的人，CD3-细胞基于CD14和CD16表达然后门。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/54255fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54255/54255fig2.jpg" /> 图2:从代表性的艾滋病毒和艾滋病病毒阳性血液样本总单核细胞表面的Glut1表达的分析 ，从HIV感染和艾滋病病毒感染初次接受治疗的人被染色用FITC标记的同型的控制或的Glut1抗体CD14 +单核细胞<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/54255fig2large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 图3:从代表性的艾滋病毒和艾滋病病毒阳性的血液样本的单核细胞亚群细胞表面的Glut1表达的分析单核细胞亚群用FITC标记的同型的控制或的Glut1抗体（A）HIV感染或（B）HIV感染治疗天真染色血液样本。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/54255fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54255/54255fig4.jpg" /> 图4:按总CD14 +单核细胞从代表艾滋病毒和艾滋病病毒阳性血样 HIV感染或感染艾滋病毒的初次接受治疗的人血液2- NBDG的摄取在终浓度与车辆或2 NBDG培养1.46μM洗涤前15分钟，并在图1描述的与细胞表面的抗体门单核细胞培养<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/54255fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54255/54255fig5.jpg" /> 图5:2 NBDG摄取单核细胞亚群的代表艾滋病毒和艾滋病病毒+全血样本 HIV感染或感染艾滋病毒的初次接受治疗的人的血液以1.46毫米的15终浓度与车辆或2 NBDG培养分洗涤前和在图1描述的细胞表面的抗体门单核细胞亚群的培育<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/54255fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> together.within页="1"&gt; <img alt="增刊。图1" src="/files/ftp_upload/54255/Supp_Figure_1_Open_FlowJo_7.6.4_Small.jpg" /> 参考图1:流式细胞仪细胞分析软件工作区窗口<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/Supp_Figure_1_Open_FlowJo_7.6.4.png" target="_blank">，请点击此处查看或下载本补充身影。</a> <img alt="增刊。图2" src="/files/ftp_upload/54255/Supp_Figure_2_Drag_and_Drop_samples_in_FlowJo_7.6.4_Small.jpg" /> 补充图2:范例数据被拖拽到这个工作区<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/Supp_Figure_2_Drag_and_Drop_samples_in_FlowJo_7.6.4.png" target="_blank">，请点击此处查看或下载本补充身影。</a> 55 / Supp_Figure_3_Double_Click_to_open_file_Small.jpg"/&gt; 补充图3:在工作区的样品，数据085被双点击，和一个第二窗口出现表示基于前向新鲜的全血样品（FSC）和侧向散射中的三个主要的细胞群（淋巴细胞，单核细胞和嗜中性粒细胞）（SSC） 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/Supp_Figure_3_Double_Click_to_open_file.png" target="_blank">请点击此处查看或下载本补充身影。</a> <img alt="增刊。图4" src="/files/ftp_upload/54255/Supp_Figure_4_Double_click_monocytes_and_select_CD3-_population_Small.jpg" /> 补充图4:单核细胞是基于前向（FSC）和侧向散射（SSC）进行门控。人口双击了这带来了一个新的窗口。 CD3上的"X"轴和CD3阴性人群选择（门控出淋巴细胞）呈S当选。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/Supp_Figure_4_Double_click_monocytes_and_select_CD3-_population.png" target="_blank">请点击此处查看或下载本补充身影。</a> <img alt="增刊。图5" src="/files/ftp_upload/54255/Supp_Figure_5_Monocyte_subpopulations_Small.jpg" /> 参考图5:CD3 -单核细胞人口双击了该长大的，其中单核细胞亚群可根据CD16和CD14表达式定义一个新的窗口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/Supp_Figure_5_Monocyte_subpopulations.png" target="_blank">请点击此处查看或下载本补充身影。</a> <img alt="增刊。图6" src="/files/ftp_upload/54255/Supp_Figure_6_Monocyte_subpopulations_MFI_Small.jpg" /> 补充图6:Monocyte亚群被选中，的Glut1细胞表面表达（平均荧光强度:MFI）由从"加统计"窗口中选择"统计"直方图窗口，"平均"，并从"参数"中选择的Glut1获得下拉菜单。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54255/Supp_Figure_6_Monocyte_subpopulations_MFI.png" target="_blank">请点击此处查看或下载本补充身影。</a>

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

一种简单的流式细胞术方法测量全血中单核细胞亚群的葡萄糖摄取和葡萄糖转运蛋白表达免疫学和感染|视频

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.

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

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

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Nanyang Technological University

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16.4K 次观看.  Macfarlane Burnet Institute for Medical Research and Public Health. 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.

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

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.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

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

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

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.

人全血中NK细胞杀伤活性的流式细胞仪分析

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

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).

全血流式细胞术分析人单核细胞亚群的表征

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

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...