这项工作得到了 nih/NIAID (R21AI119042) 以及 nih U24 样本保留化验计划 (U24-AI118656) 的支持。

样本收集是由匹兹堡大学机构审查委员会 (IRB PRO16060443, PRO10110387) 批准的。所有实验均在安全柜内进行, 并配备适当的个人防护设备。
1. 器件制造和软光刻
注: 标准软光刻技术15、16用于创建烷微通道。
<ol><li>在10:1 的碱基和固化剂的配比中混合了该聚硅烷前驱体。</li>
	<li>在微机械加工的铝模上倒入30克的前体细胞混合物 (见图 1为尺寸) 和 20 g 的在 100 mm 培养皿上的前体混合物。 注: 培养皿用于制作支撑层的厚膜 (3 毫米)。在微流体的支撑层提供了均匀的物理表面特性。另外, 还可以使用玻璃滑梯上的一层薄薄的塑料薄膜。利用铝板上的微铣削设计制造了具有特定通道尺寸的主模。本研究采用的螺旋通道是一个8环的螺旋微通道, 其中一个入口和两个插座, 半径从8毫米增加到24毫米, 以有效的大小为基础的分离。</li>
	<li>将模具和培养皿放入真空干燥, 直到表面上没有气泡, 通常为 5-10 分钟. 使用干燥的房子真空。</li>
	<li>释放真空, 将模具和培养皿放在90摄氏度烤箱中1小时。 注: 可使用热板或其他加热工具代替烤箱。</li>
	<li>从烤箱中取出模具和培养皿, 让它在室温下冷却10分钟。</li>
	<li>小心地剪下轮廓, 用2毫米冲床在设备上打上流体通路孔。 注: 冲床的大小可以根据油管或流体导轨的大小而变化。</li>
	<li>将胶带粘附在设备的通道侧, 并将其厚膜涂上, 并用镊子小心剥离以除去灰尘。</li>
	<li>将该设备的通道侧和普通的与空气等离子和粘结的纯聚硅烷 (图 1A) 制成的支撑层。</li>
	<li>将芯片放置在50毫米 x 70 毫米玻璃上, 并将其放置在70摄氏度烤箱中, 以提高粘结强度。</li>
</ol>2. 机械通气病人的气管分泌物收集
注: 气管分泌物可以从机械通气的病人在正常例行气道护理, 使用从常规方法修改的协议, 以适应标准, 成人通气病人。样品被取消辨认并且立刻被送去处理。
<ol><li>如果表示气管吸入, 使用导管提取气道分泌物。</li>
	<li>将导管小心的中途插入气管导管, 并从预先填充的10毫升注射器中注入5毫升0.9% 无菌生理盐水。</li>
	<li>当导管完全推进, 或抵抗被满足, 吸入分泌物并收集成无菌痰收集容器。</li>
	<li>收集10毫升的气管分泌物在一个无菌的痰容器, 并把它放在冰上。立即发送样品进行处理。</li>
</ol>3. 气管分泌物样品制备
注: 所有实验必须在安全柜内进行, 并配备适当的个人防护设备。
<ol><li>使用塑料10毫升注射器 (用于10x 稀释), 分散1毫升的气道分泌物样品在9毫升磷酸盐缓冲盐水 (PBS)。用钝吸管融汇粘液样本。</li>
	<li>将稀释剂与40µm 尼龙细胞过滤器一起去除大块或血块, 这样可以阻断微流体进入孔或通道。加工后将样品放在冰上。</li>
	<li>使用100x 体积的 PBS 缓冲器分散每个样品的稀释剂, 导致1,000x 稀释悬浮。在整个离解操作过程中把样品放在冰上。</li>
</ol>4. 实验设置
注: 所有实验必须在安全柜内进行, 并配备适当的个人防护设备。
<ol><li>将该芯片与流体导轨组装, 将均匀流动应用于四螺旋微 (图 1B)。 注: 四通道通过并行化提高了吞吐量, 并优化了由此产生的悬浮液的体积和细胞密度。流体导向是设计和制造的光固化成形型3D 打印机与透明树脂。该流体导向器的入口和出口端使用女性鲁尔接头, 便于连接和在操作过程中保持稳定的密封。</li>
	<li>将1/16 英寸的男性鲁尔连接器连接到进气道、内壁 (爱德) 插座和流体导轨的外壁 (下) 出口端, 并将硅胶管连接到样品悬浮液中。</li>
	<li>向入口和插座的每一端插入钝尖, 到达样品库的底部。</li>
	<li>将蠕动泵与进油管连接。</li>
	<li>将进油管和爱德出口油管的末端放置在样品库中, 并将其出口油管的末端放置在废物库中 (图 1C, D)。</li>
	<li>将经过过滤的 PBS 的50毫升管放置在样品库中, 不含钙和镁。</li>
	<li>以低流速 (~ 1 毫升/分钟) 开始泵浦, 以使该装置成为质数。 注: 当气泡在通道中捕获时, 将通道顶部与镊子, 以稳定和消除气泡。当设备完全充满缓冲液时, 改变 PBS 管, 准备气道分泌样管。</li>
	<li>当样品放置在样品库中时, 在蠕动泵上切换, 并将流速设置为4毫升/分 (图 1C, D)。</li>
	<li>当试样体积达到指定体积 (~ 1 毫升) 时, 停止操作并断开硅胶管。 注: 该方法更有效地减少了大量稀释剂 (> 50 毫升) 成微离心管容积 (~ 1 毫升) (图 2)。</li>
</ol>5. 流式细胞仪分析
注: 比较分离方法 (微流体和裂解), 采用免疫荧光法进行流式细胞术。
<ol><li>添加异硫氰酸荧光素 (FITC)-共轭小鼠抗人 CD66b 单克隆抗体和复方阿司匹林 (APC)-共轭小鼠抗人 CD45 单克隆抗体的初始悬浮 (步骤 3.3) 和结果悬浮 (步骤 4.9) (200 µL每个抗体) 的比率为1:25 伏/v。</li>
	<li>在黑暗中孵化4摄氏度的样品30分钟。</li>
	<li>在流式细胞仪上分析两个样本以量化 PMNs。 注: FITC-APC 双阳性、CD66b 阳性和 CD45 阳性的人群被认为是中性粒细胞。恢复是由输入的总细胞数和由此产生的悬浮的比率来计算的。</li>
</ol>6. NE 释放分析
注: 用微流体和裂解法比较孤立中性粒细胞的功能, 采用 NE 测定试剂盒。
<ol><li>在检测试剂盒中添加一个 (在步骤4.9 中获得的) 到最后浓度为 50 nM 的。</li>
	<li>将样品孵化37摄氏度, 2 小时。</li>
	<li>离心机悬浮在 300 x g 为10分钟。</li>
	<li>将每个上清的10µL 转移到96井板上 (在化验试剂盒中提供)。 注:96-井微板块与平底和黑聚苯乙烯可以使用以及。</li>
	<li>在每个井中添加90µL 稀释化验缓冲器 (在化验试剂盒中提供)。</li>
	<li>添加10µL 弹性蛋白酶基底 (Z-阿拉巴马-阿拉巴马 2Rh110, 提供的化验试剂盒)。</li>
	<li>在37摄氏度孵育1.5 小时。</li>
	<li>阅读96井板使用一个荧光计在 485 nm 的激发波长和发射波长 525 nm。 注: 从流式细胞术分析中, NE 的水平除以中性粒度计数。</li>
</ol>

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作者已经在这里描述的技术申请了专利申请。

在惯性微流体中, 粒子和细胞以5、18、19、20的大小在微通道中的某一侧向位置定位。由于在弯曲微通道中, 系主任阻力和惯性升力的联合作用, 大颗粒或中性粒细胞 (> 10 µm) 位于通道内, 小颗粒、粘液骨料和小于6µm 的碎片被定位在渠道外面在某些流速情况10,11,13,14。
然而, 当使用微流体时, 分离的浓度和回收率之间存在权衡, 特别是当粒子或感兴趣细胞的分离尺寸范围与其他细胞/粒子重叠时。为了达到高浓度的排序颗粒和细胞, 分离范围需要狭窄, 需要一个微调与微观观察为每个样品运行;这限制了惯性微流体在异质生物特征识别上的应用, 如气道分泌物。
在这里, 我们介绍了一个临床气道分泌物样品制备方法的体外检测使用闭环惯性微流体 (图 1B, C)。通过聚焦出口回流粒子/电池的闭环操作实现了高浓度因子和高回收率。出水口流入废料管, 并将其出口连续送入原样品管。直径大于10µm 的细胞留在最初的样品管中, 从而增加了样品的细胞密度, 而背景流体被排除在废物管上。高稀释率允许清除背景流体, 包括碎片和粘液。同时, 无论患者样本的情况如何, 该方法均可对免疫细胞进行均匀分离和隔离。
德勤是一种广泛使用的粘液裂解缓冲剂, 它将聚糖聚合中的二硫化物键分离成粘液1、21、22的细胞含量。然而, 据报道, 德勤干扰白细胞表面抗原, 这可能影响白细胞功能2。在我们的研究中, 由于不均匀的 mucolytic 效率和过滤器堵塞, 在不同的样品中, 通过德勤分离恢复的细胞含量不一致。这对制备小体积和相对较厚的气道分泌样品的初始样品变得更为关键。另一方面, 使用微流体的方法使异构患者样本中细胞内容的一致恢复 (图 3C)。如图 3B所示, 我们可以始终将气道分泌物的细胞内容隔离在干净的缓冲区中, 而不管原始的样本状态,即血腥、顽固或水样。与传统方法相比, 所提出的方法在回收样品中的碎片较少, 减少了下游生化分析中碎片的可能干扰。
通过两种浓缩方法对 NE 释放进行了研究, 以评估捕获的中性粒细胞的功能完整性 (图 3D)。中性粒细胞通过 NE 释放23,24,25去除外来有机分子。在体外26, 常用于激活中性粒细胞。用 50 nM 微流体和 mucolytic 方法分离的细胞进行了刺激。我们用人体气道分泌物样本 (标记为 P173、P174、P176、P177、P181 和 P182) 对每种方法进行了测试。以微流体为主要样本的样品在6例中均有增加。与此相反, 只有3的经处理的样品可以诱发的 NE 活性增加后, 在该刺激。另外, mucolytic 法分离的中性粒细胞比微流体分离的中性粒细胞的基线活性高得多。这些结果表明, NE 机械可能存在化学干扰,即表面束缚抗原2、3的破坏, 这表明微流体方法是一种改进的方法, 以保持中性粒细胞功能优于同质化。建议的离解方法也以全自动和完全包含的方式运作, 尽量减少病人样本的暴露和外部环境的潜在危害。
然而, 所提出的方法需要高流速, 因此, 一个稳定的射流连接。此外, 由于蠕动循环泵的固有波动, 爱德出口尺寸必须大于输出尺寸, 否则吞吐量可能会受损。我们预计, 如果有循环泵, 可以提供更稳定的流量, 建议的方法将提供更高的吞吐量。
通过提供一种从临床气道分泌物中捕获中性粒细胞的离解协议, 本研究提出的方法将扩大呼吸道疾病的临床研究范围, 如肺炎、哮喘、囊性纤维化和慢性阻塞性肺疾病。

由于细胞被封装在气道分泌物中的大量粘液中,体外测定对白细胞的功能评估受到了阻碍。Dithiothreitol () 是最常见的裂解缓冲液, 用于分离和融汇用于细胞学分析和检测的痰液, 同时为隔离细胞提供可耐受的生存能力1,2。然而, 德勤可能干扰气道中性粒细胞的表面束缚抗原, 导致中性粒细胞功能紊乱, 如弹性蛋白酶和髓 (过氧化物) 释放2,3。因此, 对人气道中性粒细胞功能的研究很少与外周血中性粒细胞有关, 这可能无法揭示4肺的生理特性。同时, 惯性微流体已取得进展, 隔离细胞从不同的病人 biomatrices5,6。惯性升力和迪恩阻力之间的平衡根据其大小对颗粒/细胞进行调整, 允许无标签粒子分离7。我们小组以前介绍了一个样品制备方法为循环肿瘤细胞8,9, 病原体在血液8, 细胞从悬浮培养10,11, 12, 和中性粒细胞 (PMNs) 从血液13,14。
在这里, 我们引入了一个协议, 用闭环惯性微流体来制备病人呼吸道分泌物的免疫细胞, 如中性粒细胞弹性蛋白酶 (NE) 测定。这种方法提供高浓度和恢复, 特别是当细胞/粒子的横向方向有明显的重叠, 细胞/粒子的利益将被删除, 这是通常观察到的临床样本。通过将内壁 (爱德) 聚焦的大颗粒或细胞回流到输入样品管, 粒子或细胞的兴趣集中在原储层, 而带有小粘着骨料的背景流体通过废物库。尽管临床样品的非均质流体特性, 该方法从气道分泌物样品中恢复到95% 以上的中性粒细胞, 稀释1000倍于干净的盐水溶液 (~ 1 毫升)。与此相反, 裂解方法根据样品条件提供了 PMNs 回收率的范围。拟议的议定书以无标签的方式捕获白细胞, 没有物理或化学干扰, 这就提供了从临床上具有挑战性的生物特征识别中获得微妙细胞的可能性, 并具有微创程序。

<table>
	<tbody>
		<tr>
			<td>PDMS precursor</td>
			<td>Dow corning</td>
			<td>184 SIL ELAST KIT 3.9KG</td>
			<td>10:1 ratio of base and curing agent</td>
		</tr>
		<tr>
			<td>VWR gravity convection oven</td>
			<td>VWR</td>
			<td>414005-128</td>
			<td>PDMS precursor to be cured in 90 deg.</td>
		</tr>
		<tr>
			<td>100mm petri dish</td>
			<td>VWR</td>
			<td>89000-324</td>
			<td>Fabrication of PDMS Supporting layer</td>
		</tr>
		<tr>
			<td>Harris Uni-core puncher</td>
			<td>Sigma-aldrich</td>
			<td>WHAWB100076</td>
			<td>2mm diameter or other depending on the tubing size</td>
		</tr>
		<tr>
			<td>Air plasma machine</td>
			<td>Femto Science</td>
			<td>Cute</td>
			<td>Surface plasma treatment for PDMS device to bottom base.</td>
		</tr>
		<tr>
			<td>2&#8221; x 3&#8221; glass slide</td>
			<td>TED PELLA, INC.</td>
			<td>2195</td>
			<td>To support PDMS device</td>
		</tr>
		<tr>
			<td>Masterflex spooled platinum-cured silicone tubing, L/S 14</td>
			<td>Cole-Parmer</td>
			<td>EW-96410-14</td>
			<td>Tubing for microfluidics and peristlatic pump</td>
		</tr>
		<tr>
			<td>1/16 inch Luer connector, male</td>
			<td>Harvard apparatus</td>
			<td>PC2 72-1443</td>
			<td>Connector for fluid guide</td>
		</tr>
		<tr>
			<td>50mL Falcon tube</td>
			<td>Corning</td>
			<td>21008-940</td>
			<td>sample collection &#38; preparation</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>45000-446&#160;</td>
			<td>Buffer solution to dilute sample</td>
		</tr>
		<tr>
			<td>Halyard Closed suction Catheter, Elbow, 14F/ channel 4.67mm</td>
			<td>HALYARD HEALTH</td>
			<td>22113</td>
			<td>Tracheal seceation suction catheter</td>
		</tr>
		<tr>
			<td>0.9% Sterile Normal saline, 10mL pre-filled syringe</td>
			<td>BD PosiFlush</td>
			<td>NHRIC: 8290-306547</td>
			<td>For tracheal seceation collection from the patients</td>
		</tr>
		<tr>
			<td>SecurTainer&#8482; III Specimen Containers, 20mL</td>
			<td>Simport</td>
			<td>1176R36</td>
			<td>Sterile sputum (airway secretion) collection container</td>
		</tr>
		<tr>
			<td>Syringe with Luer-Lok Tip, 10mL</td>
			<td>BD</td>
			<td>BD309604</td>
			<td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td>
		</tr>
		<tr>
			<td>BD&#160; Blunt Fill Needle, with BD Luer-Lok&#160; Tip</td>
			<td>BD</td>
			<td></td>
			<td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td>
		</tr>
		<tr>
			<td>40&#181;m nylon cell strainer&#160;</td>
			<td>Falcon</td>
			<td>21008-949</td>
			<td>To remove large chunk or blood clots, which can block the microfluidics access hole or the channel.</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Masterflex L/S Digital Drive)</td>
			<td>Cole-Parmer</td>
			<td>HV-07522-30</td>
			<td>operation of microfluidics</td>
		</tr>
		<tr>
			<td>BD LSR II flow cytometer</td>
			<td>BD Bioscience</td>
			<td>LSR II flow cytometer</td>
			<td>Quantification of cell recovery ratio</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody</td>
			<td>BD Bioscience</td>
			<td>561927</td>
			<td>Immunostaining of neutrophils for Flow cytometer analysis</td>
		</tr>
		<tr>
			<td>Allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody</td>
			<td>BD Bioscience</td>
			<td>561864</td>
			<td>Immunostaining of neutrophils for Flow cytometer analysis</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Thermo Fisher scientific</td>
			<td>Varioskan</td>
			<td>Plate reader for neutrophil elastase assay, ex485/em525</td>
		</tr>
		<tr>
			<td>Neutrophil elastase assay kit</td>
			<td>Cayman Chemical</td>
			<td>600610</td>
			<td>Neutrophil functionality assessment</td>
		</tr>
		<tr>
			<td>Fluoresbrite YG Microspheres 10.0&#181;m</td>
			<td>PolyScience, Inc.</td>
			<td>18140-2</td>
			<td>Fluorescent particles to express white blood cell trajectory in microfluidics</td>
		</tr>
	</tbody>
</table>

label-free neutrophil enrichment from patient-derived airway secretion using closed-loop inertial microfluidics

气道分泌物含有大量与免疫相关的细胞,例如, 白细胞、巨噬细胞和淋巴细胞, 可作为评估各种肺部疾病的主要资源, 用于研究和临床目的。然而, 由于患者粘液的异质性和粘性, 目前尚无可靠的离解方法, 不损害患者气道分泌物的宿主免疫细胞。在本研究中, 我们介绍了一种利用惯性微流体进行患者免疫评估的样品制备方法。无论临床样品的非均质流体特性如何, 该方法均能从气道分泌物样品中恢复95% 以上的中性粒细胞, 稀释1000倍于干净生理盐水的毫升。通过将浓缩输出流循环到初始样品库, 提供了高浓度、回收率和纯度的免疫细胞;再循环被认为是一个权衡的单一运行的基于注射器的惯性微流体操作。螺旋微流体的闭环操作提供无物理或化学干扰的白细胞, 佛波 12-酯 13-醋酸酯 (PMA) 诱导的弹性蛋白酶释放排列的中性粒细胞。

我们实现了透明的免疫细胞悬浮与 mucolysis 和微流体离解 (图 3A)。微流体离解收集了 4.40 x 105 PMNs 平均 (2.1 x 104到 5.60 x 105 PMNs, n = 6) 从气道分泌样品稀释了1000倍 (50 毫升总容量) 在1毫升清洁悬浮。与最初稀释剂相比, 94.0% PMNs (CD66b+/CD45+) 在一个小体积内被回收, 持续超过6个临床样本。然而, mucolysis 方法显示, 平均回收率为 53.5% (30.8-96.0%)。(图 3B)。
接下来, 用商业 NE 检测试剂盒作为概念证明, 对弹性蛋白酶的释放进行了研究。无外界刺激, mucolytic 法制备的样品比微流体法的样品具有更高的 NE 水平。从微流体和 mucolytic 离解方法中加入了可引发的悬浮液, 从而触发 NE 释放。我们用6种不同的患者的气道分泌物检测每种方法。免疫细胞与微流体分离提供了完整的功能, 显示了更多的 NE 释放的由 PMA 刺激。另一方面, mucolytic 离解法制备的细胞显示 NE 释放的不一致增加 (图 3C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57673/57673fig1.jpg"> 图 1: 螺旋微流控装置和实验设置.照片 (A) 螺旋微流体和 (B) 装置与射流适配器组装。(C) 采用螺旋惯性微流体的闭环分离原理示意图。通过将颗粒/细胞浓缩液回流回原样品库, 免疫相关细胞集中在小体积内, 而含有粘蛋白聚集物的背景流体和小红细胞连续通过连接到输出插座的废物管中取出。(D) 实验设置和10µm 粒子的荧光图像, 轨迹 (绿色) 和螺旋通道 (右上角) 的出口。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57673/57673fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57673/57673fig2.jpg"> 图 2: 采用闭环惯性微流体的气道分泌物样品制备原理图.临床诱导气道分泌物机械分散在1,000x 容积的缓冲液中。稀释剂由微流体浓缩, 产生1毫升的细胞悬浮, 具有明显的背景。适用于龙等人的许可, 版权所有 (2017) 美国化学学会17。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57673/57673fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57673/57673fig3.jpg"> 图 3: mucolytic 法与微流控分离法的代表性比较.(A) 微流控分离后气管分泌物样本 (左) 和显微图像。(B) 原始和由此产生的悬浮物的照相图像。(C) 比较 mucolytic 和微流体方法之间的中性粒细胞回收率。(D) 微流体和德勤 mucolytic 方法对分离中性粒细胞 NE 释放的比较。从微流控分离 (p < 0.0001) 的样品中, NE 释放明显触发。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57673/57673fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics at JoVE.com

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

本研究以螺旋惯性微流体闭环操作为例, 对临床气道分泌物无标签的中性粒细胞分离方法进行了论证。该方法将扩大临床对各种呼吸道疾病的体外测定。

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

label-free-neutrophil-enrichment-from-patient-derived-airway

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.3K 次观看.  Massachusetts Institute of Technology. 本研究以螺旋惯性微流体闭环操作为例, 对临床气道分泌物无标签的中性粒细胞分离方法进行了论证。该方法将扩大临床对各种呼吸道疾病的体外测定。

视频: Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

科研

免疫与感染

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

用三维定量相位成像和机器学习技术对淋巴细胞亚型进行无标签鉴定

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.