我们要感谢David Rodriguez在图形制备和精确病理学服务（德克萨斯州圣安东尼奥）使用其Navios EX流式细胞仪方面的帮助。

所有程序都需要使用适当的个人防护设备来完成。
1. TCPP储备溶液的制备，1.0 mg/ mL
注意:这可以每月准备一次。
<ol><li>使用分析天平、刮刀和称量纸，称取 49.0-50.9 mg TCPP。将重量四舍五入为 1/10 毫克。将测量的TCPP量放在避光的地方。 注意:如果重量读数不稳定，请使用静态喷枪。</li>
	<li>根据步骤1.1中称量的TCPP量，从 表1 中确定所需的纯化水和异丙醇（IPA）的量。将纯净水和异丙醇加入 100 mL 玻璃烧杯中，并覆盖封口膜以防止蒸发。</li>
	<li>根据步骤1.1中称量的TCPP量，确定 表1 中碳酸氢钠的所需量。</li>
	<li>使用分析天平、刮刀和称量纸，称取步骤1.3中测定的所需碳酸氢钠量。将重量四舍五入为 1/10 毫克。 注意:如果重量读数不稳定，请使用静态喷枪。</li>
	<li>将碳酸氢钠加入装有纯净水和异丙醇的 100 mL 烧杯中。用封口膜覆盖溶液以防止蒸发。</li>
	<li>将步骤1.5中的溶液放在搅拌板上，搅拌直至溶解（约10分钟）</li>
	<li>测量pH以确保步骤1.6中的含碳酸氢钠溶液的pH值在9和10之间。</li>
	<li>将步骤1.1中称量的TCPP缓慢加入步骤1.7的溶液中，并继续搅拌直至溶解（~30分钟）。在此步骤中避光。</li>
	<li>室温下储存在玻璃或聚丙烯容器中，避光。</li>
</ol>2.制备2-（N -吗啉基）-乙磺酸（MES）和半钠盐缓冲溶液，0.1 M，pH 6.0-6.2（"MES缓冲液"）
注意:这必须在使用当天准备并在室温下保存。
<ol><li>称出 2.50 克 MES 半钠盐，并将其加入 150 mL 塑料瓶中。</li>
	<li>加入 121 mL 纯净水，手动摇动溶解，直至看不到固体。</li>
	<li>测量MES缓冲液的pH值，确保其介于6和6.2之间。</li>
	<li>保持在室温下，以便当天使用。</li>
</ol>3. N-（3-二甲基氨基丙基）-N'-乙基碳二亚胺（EDC）粉末
<ol><li>将EDC粉末从冰箱中取出，在室温下静置，直到在步骤5中使用。</li>
</ol>4. 胺官能化聚苯乙烯珠与TCPP溶液的结合
<ol><li>将步骤 2 中制备的 4.3 mL 0.1 M MES 缓冲溶液加入 15 mL 聚丙烯管中。</li>
	<li>以最大速度涡旋胺官能化的聚苯乙烯珠悬浮液（10 μm，2.5% w/v）60 s。</li>
	<li>将 288 μL 这种新鲜涡旋的珠悬浮液添加到步骤 4.1 中的 MES 缓冲液中。</li>
	<li>以最大速度涡旋MES /珠子溶液15秒。</li>
	<li>以最大速度涡旋步骤1中制备的1 mg / mL TCPP溶液60秒。</li>
	<li>将 1.20 mL 这种新鲜涡旋的 TCPP 储备溶液添加到步骤 4.4 中的 MES/珠悬浮液中。</li>
	<li>以最大速度涡旋MES / 珠/ TCPP悬浮液15秒。</li>
	<li>在制备EDC溶液时用箔纸覆盖管子。</li>
</ol>5. N-（3-二甲基氨基丙基）-N'-乙基碳二亚胺（EDC）氢化胆酰（HCl）储备溶液的制备
注意:EDC溶液易腐烂，应在制备后立即使用。
<ol><li>将 20.0 mL 纯化水加入新的 50 mL 锥形管中。</li>
	<li>称取步骤3中的200mgEDC HCl，并将其加入水中（步骤5.1）。</li>
	<li>以最大速度涡旋EDC HCL15秒以产生澄清溶液。</li>
</ol>6. EDC HCl/MES工作溶液的制备
注意:EDC HCl/MES溶液易腐烂，应在制备后立即使用。
<ol><li>将 54.0 mL MES 缓冲溶液（在步骤 2 中制备）加入 150 mL 塑料瓶中。</li>
	<li>将 6.0 mL 的 EDC HCl 储备溶液（在步骤 5 中制备）加入 MES 缓冲溶液中，并通过振荡混合 10 秒。</li>
</ol>7. 用 TCPP 标记珠子
<ol><li>将 4.5 mL EDC 工作溶液（来自步骤 6）添加到含有磁珠和 TCPP 的 15 mL 聚丙烯管中（步骤 4.7）。</li>
	<li>将管子在室温下以35rpm的速度放入反转旋转器中16小时，并避光。</li>
	<li>将管在室温下以1，000× g离心10分钟。</li>
	<li>吸出上清液，并将珠子重悬于0.8mL汉克斯平衡盐溶液（HBSS）中。</li>
	<li>用1mL移液管将珠子溶液转移到琥珀色聚丙烯小瓶中，并储存在4°C直至进一步使用。 注意:此时珠子至少稳定 3 个月。</li>
</ol>8. 通过流式细胞术对TCPP磁珠进行质量检查（QC）
注意:QC应集中在TCPP磁珠的中位荧光强度（MFI）是否足够明亮，适合其预期用途以及该程序产生的颗粒量。有关更多详细信息，请参阅代表性结果部分。
<ol><li>将一个 5 mL 聚苯乙烯管标记为"TCPP 负磁珠"。 注意:负极珠与用于标记的胺功能化珠不同。请参阅 材料表。</li>
	<li>将另一个试管标记为"TCPP 阳性珠子"。</li>
	<li>将另一个管子标记为"彩虹珠"。</li>
	<li>将 300 μL 冰冷的 HBSS 等分到标有"TCPP 负磁珠"和"TCPP 阳性珠"的试管中。</li>
	<li>将 500 μL 冰冷的 HBSS 加入标有"彩虹珠"的试管中。</li>
	<li>以最大速度（2-3 s）短暂涡旋非官能化的聚苯乙烯（未标记）珠悬浮液，并将其加入标有"TCPP负珠"的试管中。</li>
	<li>以最大速度短暂涡旋TCPP标记的磁珠悬浮液（在步骤7.5中完成），并将其加入3μL到"TCPP阳性珠"管中。</li>
	<li>以最大速度短暂涡旋彩虹珠悬浮液，并将其滴入"彩虹珠"管中。</li>
	<li>将所有管子放在冰上，盖上盖子，避光。</li>
	<li>启动流式细胞仪的适当日常启动程序，并执行QC以验证最佳液体和激光对准。 注意:对于协议的这一部分，假设操作员接受过使用可用流式细胞仪的培训，包括标准化光散射和荧光强度的程序，以及计算正确补偿基质的基本原理。</li>
	<li>运行彩虹珠和TCPP磁珠，而不更改不同运行之间的电压设置。<ol><li>运行并收集 10，000 个彩虹珠事件。</li>
		<li>用水冲洗，并收集 10，000 个 TCPP 阴性珠子事件。</li>
		<li>用水冲洗，并收集 10，000 个 TCPP 阳性珠子的事件。</li>
		<li>进行1分钟的水冲洗。 注意:运行TCPP磁珠后用水冲洗很重要。如果没有从细胞仪中的管线上冲洗TCPP，则残留的TCPP可能会标记下一个要获取的管中的细胞。</li>
		<li>根据制造商对细胞仪的说明执行适当的清洁和关闭方案。 注意:有关代表性结果，请参见 图1。</li>
	</ol></li>
</ol>9. 珠子过滤
注意:如果流式细胞术（步骤8）的磁珠QC显示高比例的颗粒（70%或更高），请考虑使用以下方案过滤磁珠悬浮液（图2）。
<ol><li>将 3.20 mL 冰冷的 HBSS 加入步骤 7.5 中完成的 0.8 mL TCPP 珠悬浮液中（产生五倍稀释度）。</li>
	<li>以最大速度涡旋稀释的珠悬浮液15秒。</li>
	<li>从一次性 5 mL 注射器中取出柱塞。</li>
	<li>用玻璃纤维尖端过滤器（5μm，直径13mm）安装注射器。</li>
	<li>向注射器中加入4mL HBSS。</li>
	<li>加入0.5mL涡旋稀释珠悬浮液（步骤9.2）。</li>
	<li>使用柱塞通过注射器/过滤器设置以大约 2 滴/秒的速度过滤悬浮液。</li>
	<li>通过过滤器以大约 2 滴/秒的速度将 5 mL 新鲜 HBSS 吸入注射器中来洗涤珠子。</li>
	<li>以大约 2 滴/秒的速度再次将 HBSS 推入废物容器中。</li>
	<li>要从过滤器中取出珠子，请通过过滤器将另外 5 mL 新鲜 HBSS 吸入注射器中。</li>
	<li>小心地从注射器中取出过滤器。</li>
	<li>将珠悬浮液从注射器中弹出到 50 mL 锥形离心管中。</li>
	<li>将过滤器放回注射器上，再重复步骤9.10-9.12四次。然后丢弃过滤器和注射器。</li>
	<li>重复步骤9.2-9.13，直到步骤9.1中的所有珠子都被过滤掉。每次使用新鲜注射器和过滤器。</li>
	<li>在室温下以1，000× g 离心过滤的珠悬浮液10分钟。</li>
	<li>吸出每个 50 mL 管的上清液，轻轻重悬磁珠，将它们合并到 0.5 mL 新鲜 HBSS 中。</li>
	<li>用p1，000微量移液器将珠子转移到新的琥珀色，玻璃或聚丙烯小瓶中，并储存在4°C。</li>
	<li>重复步骤8以确定过滤后的珠悬浮液中TCPP相关颗粒的比例是否降低。有关代表性结果，请参见 图3。</li>
</ol>

流式细胞术中的卟啉修饰微珠

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所有作者都是bioAffinity Technologies的员工。

尽管卟啉在癌症诊断和治疗学中有许多应用2，但关于它们作为流式细胞术试剂用于鉴定原代人体组织中癌性与非癌性细胞群的潜在用途的文献有限24，25，26。我们对人痰24，27 的流式细胞术分析的研究需要用TCPP和其他几种荧光团对解离的痰细胞进行染色。然而，TCPP在指定用于其他荧光团的检测通道中的荧光溢出意味着需要TCPP标记的微球作为补偿对照。然而，没有这样的珠子是市售的，必须开发。已经报道了四芳基卟啉与二氧化硅分子筛28，29 的附着。然而，基于二氧化硅的颗粒被证明不适合制备流式细胞术标准品（数据未显示）。因此，我们的研究集中在聚苯乙烯珠上。
异丙醇-水（1:1 v/v）30中的TCPP溶液在pH 6下标记聚苯乙烯珠，但在我们的测定中，MFI相对于染色细胞太低。我们探索了共价珠修饰作为增加MFI的一种方式。Kabe等人先前使用非商业的2μm直径珠31描述了胺官能化乳胶珠与TCPP的NHS酯通过酰胺化进行共价修饰。在Kabe等人的研究中23，将TCPP与N-羟基琥珀酰亚胺（NHS）32在DMF中孵育5小时，以制备TCPP的活化NHS单酯。然后将该活化的酯溶液与含有末端胺接头的珠子反应。然而，当我们将该方案应用于商业胺官能化聚苯乙烯微珠时，标记导致微珠的MFI亮度不足以补偿。因此，我们探索了不使用NHS酯中间体的胺聚苯乙烯珠的直接酰胺化。
在这项工作中，在酰胺偶联试剂EDC存在下用TCPP处理胺官能化的聚苯乙烯珠，以在珠子上的氨基和TCPP上的羧基之间产生酰胺键。之所以选择EDC，是因为其水溶性、在宽pH范围内的水酰胺化反应中被证明的实用性、低成本和水溶性副产物33，34。我们将直径为 8.6 μm、10.6 μm 和 20 μm 的胺聚苯乙烯珠与 TCP 和 EDC 在 pH 6 的水中反应。早期的研究表明，TCPP在该pH值下仍然可溶，并且该pH值也与EDC33相容。标记的 8.6 μm 磁珠导致 MFI 对于我们的测定来说太低。20 μm磁珠具有足够高的MFI，但容易从悬浮液中沉淀并堵塞流式细胞仪（未显示）。10.6 μm磁珠具有足够的MFI，悬浮时间足够长以进行流式细胞术分析，因此被优化为补偿标准品。值得注意的是，等效的未标记磁珠具有低背景荧光，可用作阴性对照珠进行补偿。
增加EDC与TCPP的体积试剂比，同时保持磁珠数量恒定，增加磁珠MFI，直到达到约2的比率平台。在保持EDC与TCPP比率恒定的同时，TCPP与磁珠的比率增加，也观察到MFI平台。由于TCPP在没有EDC的情况下标记磁珠，因此MFI平台可能是磁珠表面上共价和非共价结合位点饱和的结果。当EDC和TCPP在pH 6下结合时，观察到与TCPP相关的颗粒副产物。该颗粒具有类似于TCPP的荧光光谱，但在没有EDC的情况下不会形成，这表明它是EDC与TCPP反应的不溶性副产物。颗粒形状不规则，虽然通常小于5μm，但可以形成更大的聚集体，并且还可以粘附在珠子的表面上。当胺聚苯乙烯珠与TCPP和EDC反应时，也观察到与TCPP相关的颗粒。颗粒量随着EDC与TCPP比的提高和TCPP与珠子比的提高而增加。最终方案中选择的试剂比例是磁珠标记程序成功的关键，因为它们可确保所得磁珠悬浮液具有高MFI，同时颗粒水平仍可接受。本协议中产生的颗粒水平并不排除使用TCPP磁珠进行补偿，但更高水平的颗粒会妨碍补偿设置的准确性35。我们还开发了一种过滤程序，如果颗粒物含量高，可以显着降低颗粒水平。
我们使用旋转搅拌器进行反应。不搅拌反应会导致染色速度变慢（数据未显示）。建议反应时间为16小时。较长的反应时间（>24小时）导致更高水平的TCPP颗粒，而较短的反应时间（<8小时）导致较低的磁珠MFI。对于未标记磁珠对照，我们没有使用未用TCPP标记的胺官能化磁珠，因为未标记磁珠的自发荧光干扰了补偿基质的正确计算。相反，我们使用非功能化的聚苯乙烯珠子，其制造和尺寸相似。此外，我们将未标记和TCPP标记的珠子保存在单独的管中。在同一管中两组磁珠时，我们观察到TCPP向未标记磁珠的一些转移（数据未显示），这导致MFI右移，从而无法正确计算补偿矩阵。
总之，实现TCPP标记磁珠高MFI的关键点是染色反应期间使用的试剂比例，染色反应的时间以及未标记微球的直径。在这项工作中，上述方案提供了TCPP染色的微球，其尺寸适合流式细胞仪使用，并且具有足够的荧光以进行补偿。TCPP染色的磁珠在4°C下300天没有显着的MFI损失。TCPP磁珠的荧光光谱与溶液中的TCPP相当。此外，用TCPP标记的磁珠和染色的A549细胞计算的补偿基质在我们的测定中产生了几乎相同的痰液谱，证明了这些磁珠的实用性。
该协议描述了在Navios EX仪器上有用的TCPP补偿磁珠的制备，但这些相同的磁珠可能无法在具有不同灵敏度36和不同检测器配置20的不同仪器上工作。然而，这项工作提出了通过磁珠大小和试剂比例调节荧光的方法。TCPP只是几种在体外和体内显示出癌细胞选择性的荧光卟啉之一2，37，38，39。并非所有荧光卟啉都可以与本协议中使用的胺官能化聚苯乙烯珠结合。事实上，这些珠子的使用仅限于含有羧酸的卟啉。通过流式细胞术测量的用卟啉对人体组织样本中的不同细胞群进行差异染色，可能会深入了解癌症的早期发展、进展和预后。使用其他卟啉对人体组织样品进行染色并通过流式细胞术进行分析，使用适当的对照珠作为补偿标准品，是进一步研究的一个有吸引力的方向。

卟啉因其荧光和肿瘤靶向特性而在生物医学领域引起了多年的关注1，2，3。光动力疗法（PDT）和声动力疗法（SDT）等治疗应用需要向癌症患者全身施用卟啉，药物在肿瘤中的积累以及肿瘤局部暴露于特定波长的激光或超声波。暴露于激光或超声波会导致卟啉产生活性氧，随后细胞死亡4，5。在光动力诊断（PDD）中，卟啉荧光用于区分癌细胞和正常细胞6。在这种情况下，原卟啉 IX 是一种天然荧光卟啉，在全身或局部注射其前体 5-氨基乙酰丙酸 （5-ALA） 后积聚在肿瘤中，用于识别胃肠道间质瘤、膀胱癌和脑癌7，8。最近，探索了5-ALA治疗作为检测多发性骨髓瘤9中微小残留病的方法。我们的实验室一直在使用四芳基卟啉TCPP（5，10，15，20-四-（4-羧基苯基）-21，23H-卟啉），因为它能够选择性地染色人痰样本中的肺癌细胞和癌症相关细胞，这一特性已在基于玻片和流式细胞术诊断测定中得到利用10。
一些卟啉是双功能的，因为它们可以用作治疗和诊断剂2，11。在生物医学研究中，这种双功能卟啉用于评估它们选择性靶向和杀死癌细胞的能力如何是其结构的功能，以及它如何受到其他化合物12，13，14，15，16的影响。卟啉的细胞摄取及其细胞毒性都可以在流式细胞术平台上以高通量方式测量。荧光卟啉的吸收和发射光谱很复杂，但大多数流式细胞术平台都配备了正确识别它们的能力。荧光卟啉的吸收光谱的特征在于380-500nm范围内的强吸收带，称为Soret带。通常在 500-750 nm 范围（Q 波段）中观察到两到四个较弱的吸收带17。大多数流式细胞仪中存在的蓝色 488 nm 激光或紫色激光 （405 nm） 可以产生适当波长的光来激发卟啉。卟啉的发射光谱通常显示600-800nm范围内18的峰，这导致与异硫氰酸荧光素或藻红蛋白（PE）荧光团的光谱重叠非常小，但与其他常用的荧光团（如别藻蓝蛋白（APC））以及串联荧光团（如PE-Cy5等）有相当大的重叠。因此，在多色流式细胞术测定中使用卟啉时，单荧光团对照对于充分校正荧光在指定用于测量卟啉荧光的通道以外的通道中的溢出至关重要。
理想情况下，用于计算一组荧光团溢出基质的单荧光基团对照（也称为"补偿对照"）应由与样品相同的细胞类型组成。但是，如果开始时的样本很少，或者样本中的目标人群非常小（例如，如果想在疾病的早期阶段观察微小的残留疾病或癌细胞），则为此目的使用样本并不是最佳的。细胞的有用替代品是微球与用于分析样品的相同荧光团耦合。许多这样的珠子是市售的;这些磁珠要么用所需的荧光团预标记（预标记的荧光团特异性微球）19，20，要么可以将荧光标记的抗体附着在其上（抗体捕获珠）20，21。虽然商业补偿微球可用于许多荧光团，但此类微球不适用于卟啉，尽管它们在基础和临床研究中的使用越来越多。
除了样品保存和适当大小的阳性与阴性群体外，使用微球作为补偿对照的其他优点是易于制备、低背景荧光和随时间推移的出色稳定性22。使用磁珠作为补偿对照的潜在缺点是，磁珠上捕获的荧光抗体的发射光谱可能与用于标记细胞的相同抗体的发射光谱不同。当使用光谱流式细胞仪20时，这可能具有特别重要的意义。因此，需要在流式细胞仪上进行磁珠作为补偿对照的开发，该流式细胞仪将用于开发磁珠的测定。此外，磁珠的显影需要包括与用相同荧光染色试剂标记的细胞进行比较。
在这里，我们描述了TCPP胺官能化聚苯乙烯补偿珠的制备，其检测通道中的中位荧光强度与痰液中TCPP标记细胞的荧光强度相当，以及它们作为流式细胞术的补偿对照的用途。等效的非功能化微球的自发荧光足够低，可以用作负荧光补偿对照。此外，这些磁珠在储存中表现出近 1 年的稳定性。

<table><tbody><tr><td>Amber plastic vials, 2 mL, U- bottom, polypropylene</td><td>Research Products International&amp;nbsp;&amp;nbsp;</td><td>ZC1028-500</td><td></td></tr><tr><td>Amine-funtionalized polystyrene divinylbenzene crosslinked (PS/DVB) beads, 10.6 &amp;mu;m diameter, 2.5% w/v aqueous suspension, 3.82 x 10&lt;sup&gt;7&lt;/sup&gt; beads/mL, 7.11 x 10&lt;sup&gt;11 &lt;/sup&gt;amine groups/ bead</td><td>Spherotech</td><td>APX-100-10</td><td>Diameter spec. 8.0-12.9 um, suspension 2.5% w/v 3.82 x 10&lt;sup&gt;7&lt;/sup&gt; beads/mL, 7.11 x 10&lt;sup&gt;11&lt;/sup&gt; amine groups/ bead</td></tr><tr><td>Conical tubes, 50 mL, Falcon</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td>with appropriate rotor</td></tr><tr><td>Disposable polystyrene bottle with cap, 150 mL</td><td>Fisher Scientific</td><td>09-761-140</td><td></td></tr><tr><td>EDC (N- (3- dimethylaminopropyl)- N&#039;- ethylcarbodiimide hydrochloride), &amp;ge;98%</td><td>Sigma</td><td>03450-1G</td><td>CAS No:&amp;nbsp; 25952-53-8</td></tr><tr><td>FlowJo Single Cell Analysis Software (v10.6.1)</td><td>BD</td><td></td><td></td></tr><tr><td>Glass coverslips, 22 x 22 mm</td><td>Fisher Scientific</td><td>12-540-BP</td><td></td></tr><tr><td>Glass fiber syringe filters (Finneran, 5 &amp;micro;m, 13 mm diameter)</td><td>Thomas Scientific</td><td>1190M60</td><td></td></tr><tr><td>Glass microscope slides, 275 x 75 x 1 mm</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (HBSS)</td><td>Fisher Scientific</td><td>14-175-095</td><td></td></tr><tr><td>Isopropanol, ACS grade</td><td>Fisher Scientific</td><td>AC423830010</td><td></td></tr><tr><td>Mechanical pipette, 1 channel, 100-1000 uL with tips</td><td>Eppendorf</td><td>3123000918</td><td></td></tr><tr><td>MES (22- (N- mopholino)- N&#039;- ethanesulfonic acid, hemisodium salt</td><td>Sigma</td><td>M0164</td><td>CAS No:&amp;nbsp; 117961-21-4</td></tr><tr><td>Navios EX flow cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Olympus BX-40 microscope with DP73 camera and 40X objective with cellSens software</td><td>Olympus</td><td></td><td>or similar</td></tr><tr><td>Pasteur pipettes, glass, 5.75&quot;</td><td>Fisher Scientific</td><td>13-678-6B</td><td></td></tr><tr><td>pH meter (UB 10 Ultra Basic)</td><td>Denver Instruments</td><td></td><td></td></tr><tr><td>Pipette controller (Drummond)</td><td>Pipete.com</td><td>DP101</td><td></td></tr><tr><td>Plastic Syringe, 5 mL</td><td>Fisher Scientific</td><td>14955452</td><td></td></tr><tr><td>Polystyrene Particles (non-functionalized), SPHERO,&amp;nbsp; 2.5% w/v, 8.0-12.9 &amp;micro;m</td><td>Spherotech</td><td>PP-100-10&amp;nbsp;</td><td></td></tr><tr><td>Polypropylene tubes, 15mL, conical</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>Polystyrene tubes, round bottom&amp;nbsp;</td><td>Fisher Scientific</td><td>14-959-2A</td><td></td></tr><tr><td>Rainbow Beads (Spherotech URCP-50-2K)</td><td>Fisher Scientific</td><td>NC9207381</td><td></td></tr><tr><td>Serological pipettes, disposable - 10 mL</td><td>Fisher Scientific</td><td>07-200-574</td><td></td></tr><tr><td>Serological pipettes, disposable - 25 mL</td><td>Fisher Scientific</td><td>07-200-576</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S6014</td><td>CAS No:&amp;nbsp; 144-55-8</td></tr><tr><td>TCPP (meso-tetra(4-carboxyphenyl)porphine)&amp;nbsp; Frontier Scientific&amp;nbsp;</td><td>Fisher Scientific</td><td>50-393-68</td><td>CAS No:&amp;nbsp; 14609-54-2</td></tr><tr><td>Tecan Spark Plate Reader (or similar)</td><td>Tecan Life Sciences</td><td></td><td></td></tr><tr><td>Tube revolver/rotator</td><td>Thermo Fisher</td><td>88881001</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>2215365</td><td></td></tr></tbody></table>

porphyrin-modified beads for use as compensation controls in flow cytometry

流式细胞术可以根据荧光测量快速表征和定量不同的细胞群。首先用一种或多种荧光试剂对细胞进行染色，每种试剂都用不同的荧光分子（荧光团）进行功能化，荧光团根据其表型特征（例如细胞表面抗原表达）选择性地与细胞结合。与细胞结合的每种试剂的荧光强度可以在流式细胞仪上使用检测指定波长范围的通道进行测量。当使用多个荧光团时，来自单个荧光团的光通常会溢出到不需要的检测通道中，这需要在称为补偿的过程中对荧光强度数据进行校正。 
细胞标记实验中使用的每个荧光团都需要补偿对照颗粒，通常是与单个荧光团结合的聚合物微球。来自流式细胞仪补偿颗粒的数据用于对荧光强度测量进行校正。该协议描述了与荧光试剂内消旋-四（4-羧基苯基）卟啉（TCPP）共价官能化的聚苯乙烯补偿珠的制备和纯化及其在流式细胞术补偿中的应用。在这项工作中，用TCPP和酰胺偶联试剂EDC（N-（3-二甲氨基丙基）-N′-乙基碳二亚胺盐酸盐）在pH 6和室温下搅拌16 h处理胺官能化的聚苯乙烯珠。通过离心分离TCPP磁珠，并重悬于pH 7缓冲液中储存。与TCPP相关的颗粒物被观察到为副产品。这些颗粒的数量可以使用可选的过滤方案来减少。所得TCPP磁珠成功用于流式细胞仪，用于用多个荧光团标记的人痰细胞实验中的补偿。TCPP珠子在冰箱中储存300天后被证明是稳定的。

该协议用于磁珠的TCPP标记相对快速和有效。图1显示了通过流式细胞术确定的TCPP磁珠标记过程的代表性结果。图1A显示了彩虹珠的标准化曲线，如在用于检测TCPP的适当通道中检测到的那样。这些微球用作流式细胞仪检测TCPP的激光电压标准化的QC。图1B显示了胺官能化聚苯乙烯TCPP标记磁珠的光散射曲线。图1C显示了非功能化微球（未标记微球）的光散射曲线;我们使用这些作为负磁珠对照来计算补偿矩阵。红色矩形表示的群体（未标记的磁珠悬浮液中不存在）表示TCPP暴露于偶联试剂EDC时形成的TCPP相关颗粒。当确定磁珠的荧光强度（FI）时，应排除这种聚集体。图1D显示了由图1B中的黑栅选择的标记磁珠的FI，这显然比（未标记的）未标记磁珠的FI高几个对数（将图1D与图1E进行比较）。
颗粒的比例（即图1B中红色门指示的事件）可能因标记批次而异。对于大多数标准流式细胞术应用，颗粒含量不超过50%的微球悬浮液允许在使用TCPP的多色流式细胞术实验中正确设置补偿。但是，例如，在某些应用中，使用自动数据分析，其中更高的颗粒含量可能会干扰适当的补偿设置。在这种情况下，建议过滤（方案步骤9和图2）。图3显示了过滤对含有~65%颗粒的珠悬浮液的影响（图3A，B的左图）。过滤后，颗粒含量降至~12%。过滤的不利影响是磁珠的一些损失（未显示）和FI的轻微损失（图3C）。在考虑过滤珠悬浮液时，应考虑这些影响。降低微粒含量的微球过滤的替代方法是改变微球、TCPP 和偶联试剂 EDC 之间的比例。然而，这些比率的变化也会影响磁珠的FI。
颗粒形成、MFI 和磁珠/TCPP/EDC 比率之间的关系如图 4 和图 5 所示。图4A和图5A分别显示了标记磁珠的MFI与EDC/TCPP比率和TCPP/磁珠比率的函数关系。随着比率的增加，小额信贷机构增加，直到稳定下来。图4B和图5B中的代表性实例表明，颗粒物含量随相应增加的比例而增加。该TCPP标记方案针对最大MFI进行了优化，同时保持颗粒含量尽可能低（图4A和图5A中的箭头）。只有在标记过程中将EDC用作偶联试剂时，才会出现颗粒。仅使用磁珠和TCPP，不会形成颗粒（未显示），但是磁珠的标记导致磁珠的FI要低得多（将图6A与图6B进行比较）。我们试图用一种基于TCPP23活化的NHS（N-羟基琥珀酰亚胺）酯的方法代替这种基于EDC的偶联方法。然而，我们发现，与仅使用磁珠和TCPP相比，使用这种替代偶联方法的FI没有显着差异，这表明使用这种方法TCPP与磁珠的共价结合可以忽略不计（将图6B与图6C进行比较）。
到目前为止，所有图表中的数据都是使用直径为10.6μm的磁珠收集的。我们假设较大的磁珠可能由于表面积增加而具有更多的TCPP结合位点，因此可以显示更高的FI。 相反，我们假设具有较小表面积和较少TCPP结合位点的较小磁珠应显示较低的FI。 使用与此处描述相同的标记方案，但使用8.6 μm磁珠或20 μm磁珠（而不是10.6 μm磁珠）， 我们发现与10.6 μm磁珠相比，8.6 μm磁珠中的FI预测降低（将图6D与图6A进行比较）。20 μm磁珠显示出与我们使用的流式细胞仪的FI值（数据未显示），但这些较大的磁珠此后很快从溶液中沉淀出来，没有进一步研究。
该协议用于用TCPP标记磁珠，目的是在使用TCPP标记细胞的流式细胞术实验中具有可靠的补偿工具。过去，我们将A549细胞用于此目的24，但标记程序并不总是导致高FI。此外，即使在固定后，细胞也不能使用超过1个月。本文所述的用TCPP标记（10.6μm）磁珠的方案一致地产生明亮染色的磁珠，如图1所示。此外，当相同的TCPP标记微球在300天后通过流式细胞术进行测试时，我们没有观察到MFI和颗粒含量的显着变化（图7）。TCPP与磁珠的耦合对其荧光发射光谱几乎没有影响（图8A;将虚线黑线与实心黑线进行比较，后者代表溶液中TCPP的荧光发射光谱）。TCPP标记磁珠的荧光发射光谱与A549肺癌细胞内测量的TCPP光谱差异更大，特别是在700-725nm波长附近（图8A;黑色虚线与橙色线相比）。然而，由于能够补偿来自多荧光团标记的痰液样本中其他信号的TCPP信号，TCPP标记的磁珠与TCPP染色的A549细胞一样有效（分别比较图8B和图8C）。
这。LMD 文件可以在 FlowRepository （https://flowrepository.org） 中找到，ID 为 FR-FCM-Z6ZP（图 1）;FR-FCM-Z5UJ（图3B，C）;FR-FCM-Z6ZR（图4B）;FR-FCM-Z6ZS（图5B）;FR-FCM-Z6ZB（图6）;FR-FCM-Z6Y5（图8B）;和FR-FCM-Z6ZT（图8C）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65294/65294fig01.jpg"> 图 1:标记有 TCPP 的磁珠的质量检查 。 （A） 彩虹珠。图中是在APC荧光团特异性通道中检测到的彩虹珠的直方图。该通道也可用于检测TCPP荧光。彩虹珠用作APC激光器的QC;第一和第四个峰应落在各自括号指示的区域。（乙，四）TCPP 标记的珠子。（中，E）未标记的珠子。这个特殊的TCPP标记磁珠溶液样品包括51.9%的磁珠（ B中的黑色矩形门）。红色矩形中的事件表示在TCPP标记期间形成的颗粒。未标记的珠子溶液中不存在这种颗粒（比较 B 和 C中的红色矩形）。TCPP标记和未标记磁珠的荧光强度分别以 D 和 E表示。缩写:SSC = 侧散;FSC = 前向散射;APC = 别藻蓝蛋白;TCPP = 内消旋四（4-羧基苯基）卟啉。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65294/65294fig02.jpg"> 图 2:协议步骤 9.3-9.12 中使用的磁珠滤波方法的示意图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig02large.jpg" target="_blank">请单击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65294/65294fig03.jpg"> 图3:过滤对颗粒和荧光强度的影响。 （A）过滤前（左图）和过滤后用TCPP标记的10μm磁珠的显微镜图像（右图）。这些图像是以20倍放大拍摄的。比例尺 = 20 μm。 （B）过滤前的TCPP珠悬浮液（左图）包括25.6%的珠子（黑色矩形）和65.7%的碎片或颗粒（红色矩形）。过滤后悬浮液中珠子的比例增加到74.7%（右图;黑色矩形），碎屑减少到11.9%。（C） B中确定的磁珠的TCPP FI的直方图。左边的直方图代表过滤前的TCPP标记磁珠，FI略高于过滤后的磁珠（右边的直方图）。在两个图中，FI 为 1 × 103 的红线是为了便于比较两个直方图。缩写:SSC = 侧散;FSC = 前向散射;APC = 别藻蓝蛋白;TCPP = 内消旋四（4-羧基苯基）卟啉;FI = 荧光强度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65294/65294fig04.jpg"> 图 4:EDC/TCPP 比率、中位荧光强度 （MFI） 和颗粒形成之间的关系。 （A）介绍了磁珠标记协议中使用的EDC / TCPP比率与随后TCPP标记的磁珠的MFI之间的关系。该曲线显示了三个独立实验的平均MFI，±标准偏差（SD）。红色箭头（3.75）表示本协议中使用的EDC和TCPP溶液的体积比。（B） 用每个曲线上方所示的 EDC/TCPP 比率制备的珠悬浮液的代表性光散射曲线。所有配置文件都来自同一实验。珠子由黑色矩形表示。颗粒形成（由红色矩形表示）随着EDC/TCPP比率的增加而增加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65294/65294fig05.jpg"> 图5.TCPP/磁珠比、MFI 和颗粒形成之间的关系。 （A）介绍了磁珠标记协议中使用的TCPP/磁珠体积比与随后的TCPP标记磁珠的MFI之间的关系。MFI迅速增加，在TCPP/磁珠比为2后达到平台期。该曲线显示了三个独立实验的平均MFI，±标准偏差（SD）。红色箭头（4.17）表示此协议中使用的比率。（B） 用每个曲线上方所示的 TCCP/磁珠比率制备的磁珠悬浮液的代表性光散射曲线。所有配置文件都来自同一实验。珠子由黑色矩形表示。颗粒形成（由红色矩形表示）随着TCPP/珠比的增加而增加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65294/65294fig06.jpg"> 图 6:耦合方法和所用磁珠尺寸的比较。 图中是用TCPP标记的磁珠荧光强度的直方图。（A）根据提供的方案标记10.6μm磁珠，包括使用偶联试剂EDC。（B）与（A）相同，但在标记协议期间省略了EDC。（C）根据Kabe等人描述的方法，使用NHS酯标记10.6μm磁珠23。（D）与（A）相同，但使用8.6μm磁珠代替10.6μm磁珠。每个轮廓中的垂直红线任意设置为 1 × 103 ，以方便直方图之间的比较。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65294/65294fig07.jpg"> 图 7:TCPP 磁珠随时间推移的稳定性。 根据方案标记TCPP标记的珠子（10μm），储存在4°C的冰箱中，并保持在黑暗中。第 0 天是标记的日子。此后的不同时间点，通过流式细胞术重新分析储存磁珠的等分试样。呈现的是相对于第 0 天值的 （A） MFI 和 （B） 颗粒百分比的测量值，该值被任意设置为 100%。水平橙色线表示高于第 0 天 10% 的值和低于第 0 天值 10% 的值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig07large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65294/65294fig08.jpg"> 图 8:TCPP 磁珠与作为补偿工具的 TCPP 染色细胞的比较。 （A）在酶标仪上测量溶液中TCCP的荧光发射光谱，TCPP与磁珠偶联以及A549肺癌细胞染色后的TCPP。可溶性和珠结合TCPP的发射光谱非常相似。两者都不同于700nm波长附近的标记A549细胞。（B，C）。将痰液样本处理成单细胞并用多种荧光团标记:AF488（FL1），BV510（FL10），PE（FL2），PE-CF594（FL3）和TCPP（FL6）。对于每种荧光团，我们制备了含有珠子与相关荧光团偶联的单色对照管。对于TCPP，设置了两个对照管:一个带有TCPP标记的磁珠，另一个具有用TCPP标记的固定A549细胞。这使我们能够创建两个补偿矩阵:（B）一个使用TCPP标记的磁珠作为TCPP（FL-6）的补偿对照，以及（C）一个使用TCPP标记的A549细胞。这两个补偿矩阵非常相似，TCPP（FL6）和FL-3之间的TCPP相关补偿差异最大。显示这些特定参数的随附点图显示几乎相同的配置文件。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/65294fig08large.jpg" target="_blank">请点击此处查看此图的大图。</a>
表1:TCPP储备溶液所需的试剂。 NaHCO3、纯净水和异丙醇（IPA）的量基于步骤1.1中称量的TCPP量（49.0mg和50.9mg之间）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65294/Bauta_etal_Table1.xlsx" target="_blank">请按此下载此表格。</a>

Watch this Scientific Journal Video about Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry at JoVE.com

作者聚焦:卟啉修饰的微珠，用作流式细胞术中的补偿对照  

该方案描述了如何通过胺官能化聚苯乙烯珠与卟啉TCPP和酰胺偶联试剂EDC的反应来制备用于流式细胞术的基于卟啉的补偿珠。过滤程序用于减少颗粒副产物。

卟啉修饰微球用作流式细胞术补偿质控

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or ...

我们开发了用卟啉TCPP修饰的聚苯乙烯微珠作为补偿对照和多色流式细胞术。该方案中使用的磁珠具有与TCPP染色细胞相当的荧光强度，并且仍然通过流式细胞仪。我们做到了这一点，目前正在研究更好的方法来去除颗粒副产物。卟啉修饰的补偿珠优于TCPP染色细胞作为补偿对照。这些速度在数月内保持稳定，没有明显的荧光损失。已知卟啉选择性地染色癌细胞。该研究将卟啉的选择性与定量流式细胞术相结合，作为人类样品高通量分析的强大平台。

porphyrin-modified-beads-for-use-as-compensation-controls-flow

Nanyang Technological University

Research

JoVE Journal

Chemistry

Porphyrin-Modified Beads for Use as Compensation Controls in Flow Cytometry

2.0K 次观看.  bioAffinity Technologies. 我们开发了用卟啉TCPP修饰的聚苯乙烯微珠作为补偿对照和多色流式细胞术。该方案中使用的磁珠具有与TCPP染色细胞相当的荧光强度，并且仍然通过流式细胞仪。我们做到了这一点，目前正在研究更好的方法来去除颗粒副产物。卟啉修饰的补偿珠优于TCPP染色细胞作为补偿对照。这些速度在数月内保持稳定，没有明显的荧光损失。已知卟啉选择性地染色癌细胞。该研...

视频: 作者聚焦:卟啉修饰的微珠，用作流式细胞术中的补偿对照  

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

科研

癌症研究

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

发育生物学

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

生物工程

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

免疫与感染

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

数据库引导流式细胞术评价骨髓细胞成熟

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

医学

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

用于高参数组织成像的免疫荧光显微镜检查和分析

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

作者聚焦：用于神经科学研究的增强型多重免疫荧光显微镜实验方案

Described here is a method that can be used to image five or more fluorescent parameters by immunofluorescent microscopy. An analysis pipeline for extracting single cells from these images and conducting single-cell analysis through flow cytometry-like gating strategies is outlined, which can identify cell subsets in tissue sections.

使用 Histoflow 流式细胞术生成和分析高参数组织学图像

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically ...

卟啉修饰微球用作流式细胞术补偿质控

副本

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此视频中的章节

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

高维流式细胞术用于解剖种植体组织的免疫功能分析

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

数据库引导流式细胞术评价骨髓细胞成熟

鼠心围细胞的分离与纯化

使用 Histoflow 流式细胞术生成和分析高参数组织学图像

使用 Histoflow 流式细胞术生成和分析高参数组织学图像