作者要感谢凯莱一个贝纳维德斯的技术贡献。这项工作是由美国心脏协会13PRE16390001（SR），美国国立卫生研究院T32HL07918（PAK），美国国立卫生研究院T32HL007457（TM），P30DK056336（BKC）的支持，NIDDK糖尿病并发症联盟（DiaComp，www.diacomp.org）授予DK076169（subaward VDU）而奥布莱恩中心P30 DK079337（VDU）。

在这个手稿的血液采集，分离和分析中描述的所有协议已审阅及批准机构审查委员会在阿拉巴马大学伯明翰分校。 1。细胞分离（从的Histopaque Sigma-Aldrich公司协议1119号及美天旎微珠阳性和阴性选择协议修改） <ol><li>离心全血在500 XG在与吊桶式转子（加速度= 5-6，并且没有刹车）离心15分钟（收集在ACD或EDTA管）。 </li><li>删除包含富含血小板的血浆（PRP）用移液管，直到1厘米留在细胞层（红血球/血沉棕黄层）上面的顶层。抛开PRP在室温下处理后。 </li><li>血沉棕黄层转移到一个无菌的50ml锥形管中，稀释至24毫升，基底的RPMI或到至少4倍的起始血沉棕黄层体积。 注意：RBC层的上半部分可以ħ等皆被包括在内，虽然这将导致成比例地增大体积的RPMI。 </li><li>使用低密度聚蔗糖为1.077（1077），用于在底层中3的15毫升锥形管中（每3ml）的比重为顶部层和高密度聚蔗糖为1.119（1119）的比重制备密度梯度。若要执行此步骤，先加1077到每个管。用5毫升的移液管狭窄添加1119到管的底部通过将移液管尖的下方1077和缓慢释放的试剂不与上部梯度混合。共6毫升密度梯度介质应该出席这个阶段分离在3 ml刻度可见阶段。 </li><li>使用自动移液管，加入8毫升稀释血（来自步骤1.3）轻轻地使用低功率设置，以防止扰乱梯度层的每个梯度管。的总体积应为14混合物在这一步。 </li><li>离心管中，在700×g离心30分钟，在室温（ACCELE配给6，无制动）。 注：在此阶段，三个不同的细胞条带应该是明显的（ 图1A）。最上面的条带（1077和等离子体之间）含有单核细胞（MNCs）和血小板，并且中频段（1007和1119之间）含有多形核细胞（中性粒细胞）和较低的频带（下文1119）中包含红细胞。 </li><li>分别用无菌玻璃移液管，而不会干扰其他细胞乐队收集跨国公司和中性粒细胞。从每个管结合的MNC人口到无菌的50ml锥形管中。重复此为中性粒细胞的人口，以及。 </li><li>新增4卷的RPMI含跨国公司和中性粒细胞分数分别稀释密度梯度每管。 </li><li>离心管中，在700×g下在室温下10分钟。 </li><li>重悬的MNC细胞沉淀，并在1ml RPMI含有0.5％的超纯无脂肪酸BSA（RPMI-BSA）的PMN细胞沉淀并转移到无菌1.5毫升管。使用台式picofuge 30沉淀细胞秒。 </li><li>弃上清，重悬的每个细胞沉淀在80微升RPMI-BSA。 </li><li>加20微升磁珠标记的-antiCD14抗体与含有MNC分数为阳性单核细胞选择的管子。对于包含PMN细胞部分管，为正选择中性粒细胞加入20微升磁珠标记 - antiCD15抗体。孵育各细胞悬浮液15分钟，在4℃。 </li><li>用1毫升的RPMI-BSA媒体洗各细胞悬浮并如前沉淀，重悬每个粒料中加入500μl的RPMI-BSA。 </li><li>标记的细胞是分离的磁性活化细胞分选（MACS）分离器的磁场。通过将柱的MACS分离器和洗涤加入细胞悬浮液前至3ml RPMI-BSA的制备MACS LS柱（每个细胞悬浮液）。 </li><li>应用细胞悬浮液后，每洗柱3次用3毫升的RPMI-BSA媒体（请务必让媒体德雷酒店Ñ​​彻底清洗之间）。收集的细胞悬浮液的流通和柱洗涤到无菌试管中。 </li><li> 以分离单核细胞和嗜中性粒细胞，从磁场删除的列的最后一次洗涤后和洗脱的阳性选择的细胞向无菌试管用5ml RPMI-BSA的使用列柱塞。 </li><li> 以分离的淋巴细胞沉淀跨国公司的从步骤1.15，在300×g离心10分钟流过的洗涤级分。弃上清，重悬细胞沉淀在80微升RPMI-BSA。加入20微升CD61和CD235a抗体孵育细胞悬液15分钟，在4°C。重复的MACS分离和以前一样，并收集虽然含有淋巴细胞的流动。 </li><li>以沉淀的单核细胞，嗜中性粒细胞和淋巴细胞分数，离心机和以前一样（步骤1.9）并丢弃上清液。细胞沉淀，应于1毫升细胞外磁介质（XF-DMEM）进行计数来悬浮。&lt;BR /&gt;注：10毫升全血应导致1-5×10 6的单核细胞/ ml，而5-20×10 6淋巴细胞和中性粒细胞/毫升。 </li><li> 以 1500×g离心在室温下8-10分钟分离的血小板 ，富血小板血浆离心机（步骤1.2）。除去血浆，用灭菌的5毫升的PBS补充有1微克/毫升PGI 2，repellet洗涤细胞沉淀一次，并悬浮最终沉淀在1ml的PBS-PGI 2缓冲区。 </li><li>确定通过比浊法血小板计数一次的血小板在PBS-PGI 2缓冲器通过分光光度计所描述的Walkowiak 等暂停使用下面的公式：[6.23 /（2.016 -腹肌800）] - 3.09×稀释因子=＃×10 8。血小板/ ml 12。 </li></ol> 2。细胞电镀<ol><li>制备细胞-德（细胞粘附）：加入90微升细胞粘附到180微升DIH 2 0，540微升的0.1N氢氧化钠双向碳酸盐（pH8.0）中，并用1N的NaOH调节pH值至7.2-7.8。用30微升准备细胞粘附的涂层以及各准备XF板（24孔，2013微孔板）。 注：细胞粘附应准备新鲜每次使用。 </li><li>培养板在37℃保持20-30分钟，然后吸出细胞粘附并用PBS洗涤孔两次（1毫升/孔）。 </li><li>对离体单核细胞，淋巴细胞，嗜中性粒细胞和血小板进行细胞计数，并使用XF-DMEM中，以允许25万个细胞/的接种密度以及对单核细胞，淋巴细胞，嗜中性粒细胞和携带至适当体积，和25×10 6个/孔的血小板。另外，测量氧化爆发反应，中性粒细胞可在125K细胞/孔接种。最终接种量为每细胞悬液应为200微升/孔。 </li><li>离心板在200×g离心1秒，没有刹车。不带刹车在300×g下再次旋转板180˚和离心机。 </li><li>最终带来良好第一卷UME到660微升与XF-DMEM培养和孵化在37℃，30分钟前XF检测。注：可以用来镀细胞前和试验后的显微照片，以确保有足够的电镀和排除细胞脱落。 </li></ol> 3。 24孔细胞外流量分析板的制备（XF24） <ol><li>水合物XF探头最少2小时前测定的校准解决方案。 </li><li>制备10倍的股票0.5微克/毫升寡霉素，0.6μMFCCP，并在XF-DMEM 10μM抗霉素A和负载75微升储备溶液的成在上面的顺序暗盒喷射口。如果氧化爆发测量是获得，注入10倍的股票100毫微克/毫升的PMA可以抗霉素-A后注入。 </li><li>校准水合和装载盒和执行XF检测。详细的方法进行分析和两个氧消耗量和pH值的变化的解释在先前的出版物9,13,14已经描述并且不ð这里详细iscussed。 </li><li> XF测定后，吸除最后加入50μlXF-DMEM中后测定，以防止从板细胞的损失，并添加50微升RIPA细胞裂解缓冲液和用于归一化进行蛋白测定。 </li></ol>

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VDU是海马生物科学顾问委员会的成员。

该协议代表了适合生物能学分析的方式几种常用的技巧血细胞分离编译。介绍了连续的技术是有利的，其他的隔离方法（ 即 FACS分析），为他们的分离大量的细胞在控制的媒体环境与放置在分离的细胞最小应力的能力。它有漫长的隔离，即使以最小的中断的缺点。这个协议作为基础初级血细胞从人类受试者，其可以外推到临床设置和平移研究的隔离。 MACS分离是可靠的细胞分离技术，提供了直接从全血中分离细胞的可能性，但是，这种方法需要更大量的抗体，而不是最适合作为在该方法中所描述的所有四种不同类型的细胞的分离。那里有BEEN没有证据表明，MACS分离结果在激活的阳性选择的白细胞使用我们的隔离协议。 MACS柱函数通过使用偶联至50nm的超顺磁性粒子的抗体在磁场中的标记的细胞封存。然后标记的细胞被洗脱出来的柱。正面和负面的选择在本协议执行，以确保迅速隔离和纯度。如果不足的细胞数是从分离获得或纯度是有问题，多种抗体可以通过LS柱被加入到样品中作为每个供应商的指令和第二通道，可能导致更高的纯度（ 表2）。我们实验室发现的高纯度和电镀效率利用现有的协议通过FACS分析分析8决赛细胞悬液。 胞通量分析仪具有较的加时，以监测氧的消耗和介质酸化实时能力她的电极。氧气消耗在单核细胞和中性粒细胞的氧化爆发响应，观察到的是NADPH氧化酶的影响，具体表现出抑制与DPI 8。我们已经看到在氧化爆发能力与时间相关的损失与长期隔离或延长XF检测。这个协议被设计和开发，以XF24使用，但也与XF96兼容于大约三分之一到二分之一XF24细胞接种密度（ 图3）。 在这个协议的设计，坚持为每个技术现有的协议是需要与纯粹来控制媒体条件下生物能分析修改的最佳性能。掌握技术后，这样的协议可以用来在平移和研究应用的各种各样测量的治疗策略的毒性和有效性，探索疾病的代谢特征，和氧化剂的生产由单核细胞和NEUTrophils在炎症条件。

监测免疫细胞（单核细胞，淋巴细胞，嗜中性粒细胞）和从血小板的生物能量健康已经认识了一段时间作为一个潜在的有用的诊断工具，以评估一个个体的整体健康的生物能量。有文献的一种新兴体归于多种疾病，如癌症，心血管疾病和神经变性疾病线粒体功能障碍1,2。这在临床上是重要的，因为线粒体功能障碍可以发起一系列促进促炎症信号传导途径，或导致细胞死亡的细胞事件。一些研究已经特征外周血单核细胞和血小板中，如纤维肌痛，糖尿病，脓毒性休克，和阿尔茨海默氏病3-7条件的线粒体功能。例如，最近的一项研究评估血小板的生物能作为标记线粒体功能和发现，2型diabet血小板IC患者已经减少线粒体氧耗7,8。从这些结果和其他人，很显然，单核细胞，淋巴细胞，嗜中性粒细胞和血小板，因为他们的调查系统循环，并可以反映局部和全局的代谢变化可以作为病理条件下的变化的生物能作为替代标志物。为了确定这种做法是否具有预测或诊断价值分析的高通量方法和所需的细胞制备的一致方法。 衡量白细胞和血小板线粒体功能的方法以前参与细胞的线粒体生物能学或评估的隔离完整细胞4,9。使用的细胞外磁通（XF）分析器细胞生物能评估的优点是，在细胞中的线粒体功能，可与内源性底物和呼吸参数如质子泄漏和最大呼吸建立容量可以被确定。我们和其他人已经使用此技术来显示，血小板生物能访问，单核细胞和淋巴细胞从人血液中分离可以建立和中间细胞类型8相比较。此外，这两种嗜中性粒细胞和单核细胞具有一氧化猝发能力，其中NAPDH氧化酶被激活并消耗氧以形成过氧化物。重要的是，这一途径是先天免疫的重要组成部分，是由全身性炎症调制。例如，已经表明，改变氧化猝发能力与各种自身免疫性疾病，如多发性硬化症，关节炎和复发性感染10,11相关联。目前没有可用来测量在临床样品中的氧化爆发不高通量定量测定。这是重要的，因为表征嗜中性粒细胞和单核细胞的氧化爆发的能力也可以作为数的病态的一个重要的诊断工具吉斯。 关键的技术挑战已经使用传统极谱技术耗氧量的测量，以及需要使用更灵敏的微量荧光技术时使用的贴壁细胞敏感性低。在这个实际视频中，我们描述的技术解决了这些问题。我们详细介绍了方法的单核细胞，淋巴细胞，嗜中性粒细胞和血小板与氧化猝发的单核细胞和人血液嗜中性粒细胞的生物能量学的分离，电镀和测量，该方法适用于生物能学和氧化猝发的临床评估为有研究者访问一个患者群体和取得新鲜血液样品的能力。

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			<td height="60" style="height:60px;width:215px;">QuadroMACS Starting Kit incl. QuadroMACS Separator and MACS MultiStand</td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-094-833</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;"><a name="RANGE!A3">CD235a (Glycophorin A) MicroBeads, human</a></td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-050-501</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">CD61 MicroBeads, human&#160; for platelets</td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-051-101</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">CD14 MicroBeads, human&#160; for monocytes</td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-050-201</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">CD15 MicroBeads, human&#160; for granulocytes</td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-046-601</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">LS Columns</td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-042-401</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">BD Vacutainer ACD Blood collection tubes</td>
			<td style="width:215px;">Fisher</td>
			<td style="width:215px;">02-684-26</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">RPMI 1640</td>
			<td style="width:215px;">Gibco</td>
			<td style="width:215px;">11835-030</td>
			<td style="width:215px;">with L-glutamine and without phenol red</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Prostaglandin I2, sodium salt</td>
			<td style="width:215px;">Cayman</td>
			<td style="width:215px;">18220</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">1.5 ml semi-micro cuvettes</td>
			<td style="width:215px;">Phenix Research Products</td>
			<td style="width:215px;">SC-2410</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Histopaque density gradient, specific gravity 1.077</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">10771</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Histopaque density gradient, specific gravity 1.119</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">11191</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Bovine Serum Albumin Fraction V</td>
			<td style="width:215px;">Roche</td>
			<td style="width:215px;">3117405001</td>
			<td style="width:215px;">fatty-acid ultra-free</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Phorbol 12-myristate 13-acetate</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">P8139</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">XF24 FluxPak</td>
			<td style="width:215px;">Seahorse Biosciences</td>
			<td style="width:215px;">100850-001</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">DMEM</td>
			<td style="width:215px;">Fisher</td>
			<td style="width:215px;">MT90113PB</td>
			<td style="width:215px;">w/o Glucose, L-Glutamine, Pyruvate, Phenol Red, and Bicarbonate</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">L-Glutamine, 200mM (100x)</td>
			<td style="width:215px;">Invitrogen</td>
			<td style="width:215px;">25030-081</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">D-Glucose</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">G7528</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Sodium Pyruvate</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">P8574</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Cell-Tak (cell adhesive)</td>
			<td style="width:215px;">BD Biosciences</td>
			<td style="width:215px;">CB-40242</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Oligomycin</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">O4876</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Antimycin A</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">A8674</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">(FCCP) Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">C-2920</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">DC Protein Assay Reagent A</td>
			<td style="width:215px;">Bio-Rad</td>
			<td style="width:215px;">500-0113</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">DC Protein Assay Reagent S</td>
			<td style="width:215px;">Bio-Rad</td>
			<td style="width:215px;">500-0015</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">DC Protein Assay Reagent B</td>
			<td style="width:215px;">Bio-Rad</td>
			<td style="width:215px;">500-0114</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;"></td>
			<td style="width:215px;"></td>
			<td style="width:215px;"></td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Equipment</td>
			<td style="width:215px;">Vendor</td>
			<td style="width:215px;">Product #</td>
			<td style="width:215px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Seahorse</td>
			<td style="width:215px;">Seahorse Biosciences</td>
			<td style="width:215px;"></td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">QuadroMACS Separator and MACS MultiStand</td>
			<td style="width:215px;">Miltenyi Biotec</td>
			<td style="width:215px;">130-090-976 and 24039</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">Spectrophotometer</td>
			<td style="width:215px;"></td>
			<td style="width:215px;"></td>
			<td style="width:215px;"></td>
		</tr>
	</tbody>
</table>

bioenergetics and the oxidative burst: protocols for the isolation and evaluation of human leukocytes and platelets

线粒体功能障碍是已知在许多病理情况，如动脉粥样硬化，糖尿病，脓毒性休克，以及神经变性疾病，但评估的变化在患者生物能量函数是具有挑战性的方面发挥显著作用。虽然疾病如糖尿病或动脉粥样硬化现在临床上使用的特定器官损伤，病理学的全身性成分，如高血糖症或炎症，可在循环中的白细胞或血小板改变生物能量函数。这个概念已经认识了一段时间，但它的广泛应用受到限制由大量需要的生物能分析原代细胞。本技术限制已经克服相结合的磁珠分离技术，细胞黏附技术，它允许贴壁细胞不活化微孔板的特殊性，以及新技术的敏感性专为高吞吐量的麦克风roplate期呼吸。这种设备的一个例子是胞外通量分析仪。这样的仪器通常使用氧和pH敏感的探针来测量变化在贴壁细胞这些参数的速率，然后可以与新陈代谢。在这里，我们详细方法的单核细胞，淋巴细胞，嗜中性粒细胞和血小板的分离和电镀，而不活化，从人血液和在这些细胞中的线粒体生物能量函数的分析。此外，我们展示了如何突发在单核细胞和嗜中性粒细胞的氧化还可以在同一样品中进行测定。因为这些方法只使用8-20毫升人类血液它们有潜力用于监测在临床环境中的活性氧的生成和生物能。

为了评估血细胞的生物能量学，全血是从人类受试者通过静脉穿刺在EDTA或ACD真空采血管采集管收集。白细胞和以下采血血小板的隔离被可视化在图1A中在协议中详述。全血样品中收集的8小时进行处理，以避免细胞死亡和激活。延长存储时间大于8小时尚未经过测试，但可能导致改变的生物能量，并在生理条件代表少。酸式柠檬酸盐葡萄糖（ACD-8毫升）和EDTA真空采血管管已用于细胞隔离与分离的细胞的生物能量功能无作用。抗凝血剂是必要的，因为凝块减少获得的细胞的数量，在聚蔗糖梯度离心以适当的相位形成的干扰，从而导致很少或几乎没有在离心分离血清获得的血小板。所有的血代表BIOLOGiCal的危害及适当的个人防护设备必须在整个即使细胞群体中分离或产生细胞裂解物的整个过程来实现。样品均按照该机构的人力废物处置标准进行处置。 将全血离心分离的富含血小板的血浆从血沉棕黄层，白色层的正上方聚集的红血细胞，其中包含的白细胞分离。血沉棕黄层施加于密度梯度离心获得外周血单核细胞（单核细胞和淋巴细胞）和多形核细胞（粒细胞）。的个人跨国公司和PMN细胞阶段红细胞污染是常见的在这个阶段，应该在MACS纯化过程中得到解决。的各种细胞类型，然后由孵化与磁性抗体获得，得到纯的馏分。单核细胞是由PBMC部分与CD14，流体可通过从吨的孵化收集他单核细胞含有淋巴细胞，然后将其耗尽红细胞和血小板。嗜中性粒细胞是由多形核细胞层用CD15抗体（ 图1A）的培养获得的。此后，血小板可通过富含血小板的血浆的离心沉淀，并洗涤以除去其它血浆组分。这些选择后的血小板，CD14 +单核细胞，淋巴细胞，CD15 +中性粒细胞进行计数并接种于细胞粘附介质涂覆XF分析器板为生物能学和氧化爆发分析。 应在无菌条件下进行该过程的每个阶段和鼓励，以防止在单核细胞和嗜中性粒细胞的氧化猝发反应的过早激活。小于隔离在无菌条件下的一个指标是显而易见的，当嗜中性粒细胞，其在基础条件下具有很少或几乎没有氧消耗，开始细胞外通量测定升高的OCR测量该逐渐增加或下降，在整个测定。我们强调使用光学显微镜在200倍或更大，以确保激活的形态没有迹象之前，测定发生的成像单元的重要性。例如，嗜中性粒细胞正常显示一个圆形不规则的形状，明显的细胞界限， 如图1B所示。然而，这些细胞就会压扁对板的表面时被激活，并失去其独特的细胞形态。之前我们已经报道活化的白细胞和血小板的形态变化使用此协议，必须采取额外的措施，以确保无菌条件下，如果遇到激活8。 该协议的一个简化方案被认为是在图2中作为一个时间表，其中的细胞分离，XF板制备和XF测定法的主要步骤是详细。隔离包括细胞类型的密度梯度分离和之后通过磁性分离。平行于细胞的分离过程中，XF板的准备是必要的，以避免电池板或开始XF检测延迟。这是关键，因为显著延误可导致细胞的活化和减少生物能量参数。 分离后不足的细胞浓度是一个普遍的问题，每个离心过程的优化是必要的，以避免细胞的损失。成功地隔离了对少至6毫升的血液被执行，但改变可以取决于循环所需要的细胞类型的浓度进行。如果密度梯度制备不当，不同细胞阶段可能不可见和细胞的损失可能发生（见参考资料中的视觉展示朱庇特的教学视频）。血小板从PRP中分离不足是罕见的，但已经发生，如果洗涤缓冲液中不包含PGI 2，或者如果需要隔离低于室温的地方。血小板一风险ctivati​​on通常防止，如果血小板是最后一个单元，以进行分离，但是，如果血小板保持在洗涤缓冲液长时间的生物能会受到影响。有过小的血小板种群的1500×g离心离心分离过程中没有沉淀，并且这些细胞的生物能量没有被广泛表征的实例。请参阅表2为一个更完整的故障排除指南。 白细胞和血小板的生物能访问可以使用XF分析仪，它测量实时O 2消耗量在细胞非侵入性地确定。每个信元类型被镀上XF24板与5个重复中所示的图3A中 ， 表1表示按细胞类型每孔测定，平均蛋白质含量在预期基础和氧化迸发值。高通量技术是可用的，如XF96，这允许4供体同时被评定为如图3B所示。测定后，将数据导出到使用适当的XF软件和Microsoft Excel工作站计算机进行分析。 OCR的值标准化为在相应的孔中的总蛋白含量，并表示为皮摩尔/ min / mg蛋白。最近报道我们小组8名不同的细胞类型的代表生物能氧化突发配置。的测定（基肥，ATP联的，质子泄漏，最大，nonmitochondrial和氧化猝发）的生物能量参数的进一步分析，可以计算出各孔如前所示及以下8所述。 基底耗氧率（OCR）是由所述第一测量值3确定为如图4A所示。寡霉素（0.5微克/毫升），线粒体ATP合酶抑制剂注入XF介质来估计OCR耦合到ATP合成和代表中<html>特德作为ATP挂钩。残余OCR减去nonmitochondrial OCR可以归因于质子泄漏。然后FCCP解偶联剂被添加，以确定最大的OCR，随后抗霉素A，线粒体呼吸的抑制剂，来确定耗氧nonmitochondrial来源。备用容量是ATP，可以有活力的需求下被制造，以及可以计算呼吸的最大速率和基底之间的差的量的量度。为了确定氧化猝发能力，佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）一个PKC激动剂注入以增加NADPH氧化酶活性，并增加氧的消耗速率以下PMA刺激可使用XF分析仪进行测定。 图4B说明如何计算线粒体功能和氧化迸发的不同参数。 <html> 1301/51301fig1highres.jpg“SRC =”/ files/ftp_upload/51301/51301fig1.jpg“/&gt; 图1：从全血中分离白细胞和血小板的。 A）新鲜标本收集通过离心分离成其血浆和细胞成分，并通过密度梯度纯化，磁性细胞分选（MACS）由正（单核细胞和中性粒细胞）或阴性选择（淋巴细胞），而血小板被分离出高一旦悬浮在XF的DMEM中和镀在指定的细胞密度分离细胞群速的富血小板血浆的离心B）图像。 <a href="https://www.jove.com/files/ftp_upload/51301/51301fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ghres.jpg“SRC =”/ files/ftp_upload/51301/51301fig2.jpg“/&gt; 图2。准备XF24板为白细胞和血小板的生物能的研究。详细通过细胞分离和电镀和墨盒水化和注射港口装卸准备一个XF24分析板的时间表。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51301/51301fig3highres.jpg" src="/files/ftp_upload/51301/51301fig3.jpg" /> 上XF24及XF96分析仪板A）白细胞（125-250K细胞/孔）和血小板（25×10 6个细胞/孔）从单一供体的白细胞和血小板的图3。布局镀在其中背景的XF24分析板控制。 75微升10倍股寡霉素（0.5微克/毫升），FCCP（0.6μM），抗霉素A（10μM），和PMA（100毫微克/毫升）稀释，在XF介质中加载所指示的喷射口B）白细胞（75-150K细胞/孔）和血小板（10×10 6个细胞/孔）从多达四个供体可以在180微升XF-DMEM中，总体积被镀上XF96。 20μl的10倍股寡霉素（1.0微克/毫升）的，FCCP（0.6μM），抗霉素A（10μM），和PMA（100毫微克/毫升）稀释，在XF-DMEM中装入所指示的喷射口。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51301/51301fig4highres.jpg" src="/files/ftp_upload/51301/51301fig4.jpg" /> 线粒体和单核细胞氧化爆发图4。生物能指标的分析 。 一）代表的耗氧率（OCR）的痕迹。基础OCR成立（由圆圈数字表示第3个评分）。然后顺序寡的增加（O，0.5微克/毫升），FCCP（F; 0.6微米）和抗霉素-A（A; 10微米）注射，以确定线粒体和非活性细胞nonmitochondrial呼吸。最后，PMA（100毫微克/毫升）加入到测量氧化爆发B）线粒体功能和氧化爆发的模块化分析，所指示的计算采用率的差异。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="66"><td height="66" style="height:67px;width:215px;"></td><td style="width:215px;">最佳播种密度</td><td style="width:215px;">平均。基础OCR（皮摩尔O 2 /分钟） </td><td style="width:215px;">平均。氧化爆发的OCR（皮摩尔O 2 /分钟） </td><td style="width:215px;">平均。蛋白（微克）/孔</td></tr><tr height="40"><td height="40" style="height:40px;">单核细胞</td><td> 25万个细胞/孔</td><td> 83.9（±13.0） </td><td> 300.3（±58.8） </td><td> 19.0（±2.4） </td></tr><tr height="40"><td height="40"风格=“高度：40PX;”&gt;中性粒细胞</td><td> 25万个细胞/孔</td><td> 6.1（±2.2） </td><td> 1411.8（±233.3） </td><td> 20.6（±2.7） </td></tr><tr height="40"><td height="40" style="height:40px;">淋巴细胞</td><td> 25万个细胞/孔</td><td> 52.9（±7.7） </td><td> 15（±4.1） </td><td> 13.2（±2.1） </td></tr><tr height="40"><td height="40" style="height:40px;">血小板</td><td> 25×10 6个细胞/孔</td><td> 199.4（±20.3） </td><td> 24（±2.7） </td><td> 47.5（±3.1） </td></tr></tbody></table> 表1中。正常参数XF24分析：平均基底（速率3）和氧化爆发（速度7）OCR无法矫正到nonmitochondrial OCR或蛋白质都表现出镀在指定的细胞密度的细胞类型。每口井的平均蛋白含量所表现出的直流Lowry法的执行。这些数据代表的平均值6-8健康献血者含±SEM括号。 <table border="0" cellpadding="0" cellspacing="0"><colgroup><col span="4" /></colgroup><tbody><tr height="66"><td>步</td><td>问题</td><td>可能的原因</td><td>解</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 1.2 </td><td style="width:199px;">清晰的等离子</td><td style="width:199px;">血小板离心沉淀过程中</td><td style="width:199px;">减少离心时间为10分钟或慢XG 400 </td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"></td><td style="width:199px;">乳白色的血浆</td><td style="width:199px;">多余的脂肪</td><td style="width:199px;">避免餐后集合</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 1.6 </td><td style="width:199px;">不完整的细胞带形成</td><td style="width:199px;">恶魔罗珀梯度准备，冷剂</td><td style="width:199px;">避免在移液，利用室温试剂干扰梯度</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"></td><td style="width:199px;">在细胞团块乐队</td><td style="width:199px;">的血液凝固可能发生</td><td style="width:199px;">使用抗凝血药，如ACD或EDTA收集血液</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 1.10 </td><td style="width:199px;">无细胞沉淀</td><td style="width:199px;">见步骤1.6 </td><td style="width:199px;">如果上清液朦胧见下文</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"></td><td style="width:199px;">朦胧的上清液</td><td style="width:199px;">沉重的聚蔗糖梯度污染，污染血小板</td><td style="width:199px;">提高离心速度为900 XG，二琵琶与更多的RPMI </td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 1.16，1.17 </td><td style="width:199px;">白细胞低收益率</td><td style="width:199px;">重红细胞污染，没有足够的全血，凝血</td><td style="width:199px;">双抗体和RPMI-BSA卷，收集更多的血，加抗凝剂</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 1.19 </td><td style="width:199px;">血小板聚集</td><td style="width:199px;"> PGI 2省略，受寒媒体，长时间贮存在血浆</td><td style="width:199px;">添加PGI 2血小板洗涤缓冲液，用室温试剂</td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 1.20 </td><td style="width:199px;">血小板计数低</td><td style="width:199px;">在初级离心，血小板聚集亏损</td><td style="width:199px;">看步骤1.2和1.19 </td></tr><tr height="66"><td height="66" style="height:67px;width:199px;"> 2.3 </td><td style="width:199px;"> RBC污染</td><td style="width:199px;"></td><td style="width:199px;">重复MACS分离</td></tr></tbody></table> 表2。白细胞和血小板隔离故障诊断。下表列出了在从全血和解决方案，可以指导用户完成故障排除过程白细胞和血小板细胞分离遇到的常见问题。

Watch this Scientific Journal Video about Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets at JoVE.com

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

血液白细胞和血小板可以作为一个单独的整体生物能量健康的一个标志，所以具有监视病理过程和治疗的影响的可能性。这里我们描述的方法来分离并测量线粒体功能和在这些细胞中的氧化爆发。

生物能量和氧化突发：协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

bioenergetics-oxidative-burst-protocols-for-isolation-evaluation

Research

JoVE Journal

Immunology and Infection

36.9K 次观看.  University of Alabama at Birmingham. 血液白细胞和血小板可以作为一个单独的整体生物能量健康的一个标志，所以具有监视病理过程和治疗的影响的可能性。这里我们描述的方法来分离并测量线粒体功能和在这些细胞中的氧化爆发。 

视频: Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

从人类母乳中分离白细胞，用于HIV靶点抗体依赖性细胞噬菌体测定

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...