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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Method of using 5,10,15,20-tetrakis(carboxyphenyl)porphine for detecting cancers of the lung Available from: <a target="_blank" href="https://www.osti.gov/deopatents/biblio/7117152">https://www.osti.gov/deopatents/biblio/7117152</a> (1992)</li><li>Grayson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality-controlled+sputum+analysis+by+flow+cytometry.">Quality-controlled sputum analysis by flow cytometry.</a> Journal of Visualized Experiments. (174), e62785 (2021).</li><li>Anjali, K., Christopher, J., Sakthivel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruthenium-based+macromolecules+as+potential+catalysts+in+homogeneous+and+heterogeneous+phases+for+the+utilization+of+carbon+dioxide.">Ruthenium-based macromolecules as potential catalysts in homogeneous and heterogeneous phases for the utilization of carbon dioxide.</a> ACS Omega. 4 (8), 13454-13464 (2019).</li><li>Yadav, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+preparation+and+applications+of+organo-functionalized+porous+materials.">Recent advances in the preparation and applications of organo-functionalized porous materials.</a> Chemistry. 15 (17), 2588-2621 (2020).</li><li>. US7670799B2 - Method for making 5,10,15,10-tetrakis (carboxyphenyl) porphine (TCPP) solutions and composition compromising TCPP Available from: <a target="_blank" href="https://patents.google.com/patent/US7670799B2/en">https://patents.google.com/patent/US7670799B2/en</a> (2023)</li><li>Shimizu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+affinity+beads+for+identifying+drug+receptors.">High-performance affinity beads for identifying drug receptors.</a> Nature Biotechnology. 18 (8), 877-881 (2000).</li><li>Anderson, G. W., Zimmerman, J. E., Callahan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+esters+of+N-hydroxysuccinimide+in+peptide+synthesis.">The use of esters of N-hydroxysuccinimide in peptide synthesis.</a> Journal of the American Chemical Society. 86 (9), 1839-1842 (1964).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. , (2013).</li><li>El-Faham, A., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+coupling+reagents,+more+than+a+letter+soup.">Peptide coupling reagents, more than a letter soup.</a> Chemical Reviews. 111 (11), 6557-6602 (2011).</li><li>Hulspas, R., O'Gorman, M. R. G., Wood, B. L., Gratama, J. W., Sutherland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+the+control+of+background+fluorescence+in+clinical+flow+cytometry.">Considerations for the control of background fluorescence in clinical flow cytometry.</a> Cytometry Part B. 76 (6), 355-364 (2009).</li><li>Hoffman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization,+calibration,+and+control+in+flow+cytometry.">Standardization, calibration, and control in flow cytometry.</a> Current Protocols in Cytometry. , (2005).</li><li>Ethirajan, M., Chen, Y., Joshi, P., Pandey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+porphyrin+chemistry+in+tumor+imaging+and+photodynamic+therapy.">The role of porphyrin chemistry in tumor imaging and photodynamic therapy.</a> Chemical Society Reviews. 40 (1), 340-362 (2011).</li><li>Beharry, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+photodynamic+therapy:+New+probes+for+cancer+imaging+and+treatment.">Next-generation photodynamic therapy: New probes for cancer imaging and treatment.</a> Biochemistry. 57 (2), 173-174 (2018).</li><li>El-Far, M., Pimstone, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+28+porphyrins+and+their+abilities+to+localize+in+mammary+mouse+carcinoma:+Uroporphyrin+I+superior+to+hematoporphyrin+derivative.">A comparative study of 28 porphyrins and their abilities to localize in mammary mouse carcinoma: Uroporphyrin I superior to hematoporphyrin derivative.</a> Progress in Clinical and Biological Research. 170, 661-672 (1984).</li></ol>

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，TCP...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amber plastic vials, 2 mL, U- bottom, polypropylene</td>
			<td>Research Products International&#160;&#160;</td>
			<td>ZC1028-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine-funtionalized polystyrene divinylbenzene crosslinked (PS/DVB) beads, 10.6 &#956;m diameter, 2.5% w/v aqueous suspension, 3.82 x 107 beads/mL, 7.11 x 1011 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 107 beads/mL, 7.11 x 1011 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&#39;- ethylcarbodiimide hydrochloride), &#8805;98%</td>
			<td>Sigma</td>
			<td>03450-1G</td>
			<td>CAS No:&#160; 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 &#181;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&#39;- ethanesulfonic acid, hemisodium salt</td>
			<td>Sigma</td>
			<td>M0164</td>
			<td>CAS No:&#160; 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&#34;</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,&#160; 2.5% w/v, 8.0-12.9 &#181;m</td>
			<td>Spherotech</td>
			<td>PP-100-10&#160;</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&#160;</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 (NaHCO3)</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>CAS No:&#160; 144-55-8</td>
		</tr>
		<tr>
			<td>TCPP (meso-tetra(4-carboxyphenyl)porphine)&#160; Frontier Scientific&#160;</td>
			<td>Fisher Scientific</td>
			<td>50-393-68</td>
			<td>CAS No:&#160; 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。

Porphyrins have been of interest for many years in the biomedical field owing to their fluorescence and tumor-targeting properties1,2,3. Therapeutic applications such as photodynamic therapy (PDT) and sonodynamic therapy (SDT) entail the systemic administration of a porphyrin to a cancer patient, the accumulation of the drug in the tumor, and the localized exposure of the tumor to a laser light of a specific wavelength or ultrasound. The exposure to laser light or ultrasound leads to the generation of reactive oxygen species by the porphyrin and subsequent cell death4,5. In photodynamic diagnosis (PDD), porphyrin fluorescence is used to distinguish cancer cells from normal cells6. In this context, protoporphyrin IX, a natural fluorescent porphyrin that accumulates in tumors upon the systemic or local injection of its precursor, 5-aminolevulinic acid (5-ALA), is used to identify gastrointestinal stromal tumors, bladder cancer, and brain cancer7,8. More recently, 5-ALA treatment was explored as an approach to detect minimal residual disease in multiple myeloma9. Our laboratory has been using the tetraaryl porphyrin TCPP (5,10,15,20-tetrakis-(4-carboxyphenyl)-21,23H-porphine) for its ability to selectively stain lung cancer cells and cancer-associated cells in human sputum samples, which is a property that has been exploited in slide-based and flow cytometric diagnostic assays10.
Some porphyrins are bifunctional in that they can be used as therapeutic and diagnostic agents2,11. In biomedical research, such bifunctional porphyrins are used to evaluate how their ability to selectively target and kill cancer cells is a function of their structure as well as how it is affected by the presence of other compounds12,13,14,15,16. Both the cellular uptake of porphyrins and their cytotoxicity can be measured on a flow cytometric platform in a high-throughput manner. The absorption and emission spectra of fluorescent porphyrins are complex, but most flow cytometric platforms are equipped to correctly identify them. The absorption spectrum of fluorescent porphyrins is characterized by a strong absorption band in the 380-500 nm range, known as the Soret band. Two to four weaker absorption bands are generally observed in the 500-750 nm range (Q bands)17. A blue&#160;488 nm laser, present in most flow cytometers, or a violet laser (405 nm) can generate light of the appropriate wavelength to excite porphyrins. The emission spectra of porphyrins typically display peaks in the 600-800 nm range18, which results in very little spectral overlap with fluorescein isothiocyanate or phycoerythrin (PE) fluorophores but considerable overlap with other often-used fluorophores, such as allophycocyanin (APC), as well as tandem fluorophores, such as PE-Cy5 and others. Therefore, when using porphyrins in multi-color flow cytometry assays, single-fluorophore controls are essential to adequately correct the spillover of fluorescence in channels other than the one designated to measure the porphyrin&#39;s fluorescence.
Ideally, the single-fluorophore controls used to calculate the spillover matrix for a panel of fluorophores (also called &#34;compensation controls&#34;) should consist of the same cell type(s) as the sample. However, using the sample for this purpose is not optimal if there is very little sample to begin with or if the target population within the sample is very small&#160;(for example, if one wants to look at minimal residual disease or cancer cells at the early stages of the disease). A useful alternative to cells is beads coupled with the same fluorophore that is used to analyze the sample. Many such beads are commercially available; these beads are either prelabeled with the desired fluorophore (prelabeled fluorophore-specific beads)19,20, or a fluorescently labeled antibody can be attached to them (antibody capture beads)20,21. While commercial compensation beads are available for many fluorophores, such beads are unavailable for porphyrins, despite their increasing use in basic and clinical research.
In addition to sample preservation and appropriately sized positive versus negative populations, the other advantages of using beads as compensation controls are the ease of preparation, low background fluorescence, and excellent stability over time22. The potential disadvantage of using beads as a compensation control is that the emission spectrum of the fluorescent antibody captured on beads may differ from that of the same antibody used to label the cells. This may be of specific importance when using a spectral flow cytometer20. Therefore, the development of beads as a compensation control needs to be performed on the flow cytometer that will be used for the assay for which the beads are developed. Moreover, the development of the beads needs to include a comparison with cells labeled with the same fluorescent staining reagent.
Here, we describe the preparation of TCPP amine-functionalized polystyrene compensation beads, whose median fluorescence intensity in the detection channel was comparable to that of TCPP-labeled cells in sputum, and their use as compensation controls for flow cytometry. The autofluorescence of equivalent, non-functionalized beads was sufficiently low for their use as negative fluorescence compensation controls. In addition, these beads demonstrated stability in storage for nearly 1 year.

作者聚焦：卟啉修饰的微珠，用作流式细胞术中的补偿对照  

卟啉修饰微球用作流式细胞术补偿质控

We developed polystyrene beads modified with the porphyrin TCPP as compensation controls and multicolor flow cytometry. The beads used in this protocol have fluorescence intensity comparable to TCPP stain cells and still passed through the flow cytometer. We accomplished that and are currently working on better methods to remove the particulate byproduct.Porphyrin-modified compensation beads were preferable to TCPP stained cells as compensation controls. These speeds were stable over months without significant loss of fluorescence. Porphyrins are known to stain cancer cells selectively.The research combines the selectivity of porphyrins with quantitative flow cytometry as a powerful platform for high-throughput analysis of human samples.

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?...

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

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。

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

Research

JoVE Journal

Chemistry

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

1.4K 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  

Flow cytometry can rapidly characterize and quantify diverse cell populations based on fluorescence measurements. The cells are first stained with one or more fluorescent reagents, each functionalized with a different fluorescent molecule (fluorophore) that binds to cells selectively based on their phenotypic characteristics, such as cell surface antigen expression. The intensity of fluorescence from each reagent bound to cells can be measured on the flow cytometer using channels that detect a specified range of wavelengths. When multiple fluorophores are used, the light from individual fluorophores often spills over into undesired detection channels, which requires a correction to the fluorescence intensity data in a process called compensation. 
Compensation control particles, typically polymer beads bound to a single fluorophore, are needed for each fluorophore used in a cell labeling experiment. Data from compensation particles from the flow cytometer are used to apply a correction to the fluorescence intensity measurements. This protocol describes the preparation and purification of polystyrene compensation beads covalently functionalized with the fluorescent reagent meso-tetra(4-carboxyphenyl) porphine (TCPP) and their application in flow cytometry compensation. In this work, amine-functionalized polystyrene beads were treated with TCPP and the amide coupling reagent EDC (N-(3-dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride) at pH 6 and at room temperature for 16 h with agitation. The TCPP beads were isolated by centrifugation and resuspended in a pH 7 buffer for storage. TCPP-related particulates were observed as a byproduct. The number of these particulates could be reduced using an optional filtration protocol. The resultant TCPP beads were successfully used on a flow cytometer for compensation in experiments with human sputum cells labeled with multiple fluorophores. The TCPP beads proved stable following storage in a refrigerator for 300 days.

The protocol describes how porphyrin-based compensation beads for flow cytometry are prepared by the reaction of amine-functionalized polystyrene beads with the porphyrin TCPP and the amide coupling reagent EDC. A filtration procedure is used to reduce the particulate byproducts.

科研

化学

Preparation of Meso-Tetra(4-Carboxyphenyl) Porphine (TCPP) Stock Solution

To begin, weigh 49.0 to 50.9 milligrams of TCPP using an analytical balance. Round the weight to 1/10 of a milligram. Set the measured amount of TCPP aside, protected from light.Based on the amount of TCPP, add the required amounts of purified water and isopropanol to a 100 milliliter glass beaker and cover it with parafilm to protect it from evaporation. Next, weigh the required amount of sodium bicarbonate based on the amount of TCPP and add it to the 100 milliliter beaker containing purified water and isopropanol. Cover the solution with parafilm to protect it from evaporation, and place it on a stir plate for approximately 10 minutes.Once the contents are dissolved, ensure that the pH of the solution is between nine and 10. Next, carefully add the weighted TCPP, protect the solution from light, and stir it for approximately 30 minutes until dissolved. Store the solution in a polypropylene container at room temperature protected from light.

内消旋-四（4-羧基苯基）卟啉（TCPP）储备溶液的制备

Combining Amine-Functionalized Polystyrene Beads with TCPP Solution

Begin by adding 4.3 milliliters of 0.1 molar MES buffer solution to a 15 milliliter polypropylene tube. Then add 288 microliters of the vortexed amine functionalized polystyrene bead suspension to the 15 milliliter polypropylene tube containing MES buffer. Vortex the MES bead solution for 15 seconds at maximum speed.Next, vortex the one milligram per milliliter TCPP solution for 60 seconds at maximum speed. Then add 1.2 milliliters of this solution to the MES bead suspension. After vortexing the resultant suspension for 15 seconds, cover the tube with foil.

胺官能化聚苯乙烯微珠与TCPP溶液的结合

Labeling Amine-Functionalized Polystyrene Beads with TCPP

To begin, add freshly prepared 4.5 milliliters of EDC working solution to a 15 milliliter polypropylene tube containing the amine-functionalized polystyrene beads and TCPP in MES buffer. Place the tube in an inverting rotator at 35 RPM for 16 hours at room temperature protected from light. Then, centrifuge it for 10 minutes.Aspirate the supernatant and re-suspend the beads in 0.8 milliliters of Hank's Balanced Salt Solution. Then, using a one milliliter pipette, transfer the bead solution to an amber polypropylene vial and store it at four degrees Celsius. The relationship between the particulate formation, the median, fluorescence intensity or MFI, and the bead TCPP/EDC ratio showed that with increasing ratios, the MFI increased until it formed a plateau.The particulates appeared only when EDC was used as a coupling reagent during the labeling procedure. Labeling the beads with TCPP alone resulted in much lower fluorescence intensity. When TCPP-labeled beads were tested after 300 days, no significant change in the MFI or in the particulate content was observed.The coupling of TCPP to the beads had a minimum effect on its fluorescence emission spectrum. However, its spectrum differed more from TCPP-labeled A549 lung cancer cells. In a multi-flora four labeled sputum sample, the TCPP-labeled beads worked equally well as the TCPP-stained A549 cells.

用TCPP标记胺功能化聚苯乙烯珠

Quality Check (QC) of the TCPP Beads by Flow Cytometry

Begin the quality check of the beads labeled as meso-tetra 4-carboxyphenyl porphine or TCPP beads in the flow cytometer by running and collecting 10, 000 events of the rainbow beads. Then perform a rinse with water and collect 10, 000 events of the TCPP negative beads. After rinsing with water again, collect 10, 000 events of the TCPP positive beads.Then perform a one minute water rinse before appropriate cleaning and shut down the flow cytometer. A representative outcome of the TCPP bead labeling process determined by flow cytometry is shown here with a standardized profile of rainbow beads. These beads serve as quality checks for standardizing the laser voltages to detect TCPP.The light scatter profile of amine functionalized polystyrene TCPP labeled beads was observed. The light scatter profile of non-functionalized beads was used as a negative bead control to calculate the compensation matrix. The population indicated by the red rectangle in the TCPP labeled beads was absent in the unlabeled bead suspension representing that the TCPP related particulates were not formed in the absence of TCPP and EDC.The fluorescence intensity of the labeled beads selected by the black gate was several logs higher than that of the unlabeled beads.

通过流式细胞术对TCPP磁珠进行质量检查（QC）

TCPP Bead Filtration

Begin the TCPP bead filtration by diluting 0.8 milliliters of the TCPP bead suspension with 3.20 milliliters of ice-cold Hanks'Balanced Salt Solution, or HBSS. Vortex the diluted bead suspension at maximum speed for 15 seconds. Then, remove the plunger from a disposable five-milliliter syringe and fit the syringe with a five-micrometer glass fiber tip filter having a 13-millimeter diameter.Next, add four milliliters of HBSS to the syringe before adding 0.5 milliliters of the vortexed diluted bead suspension. Using the plunger, filter the bead suspension through the syringe filter at approximately two drops per second. Then, wash the beads by drawing five milliliters of fresh HBSS into the syringe through the filter and pushing the HBSS out into the waste container at approximately two drops per second.To remove the beads from the filter, draw another five milliliters of fresh HBSS into the syringe through the filter. Then, remove the filter carefully from the syringe. After ejecting the bead suspension from the syringe into a 50-milliliter conical centrifuge tube, place the fresh filter back on the syringe and repeat the five-milliliter HBSS wash four times.Once done, discard the filter and syringe. Centrifuge the filtered bead suspensions at room temperature for 10 minutes at 1, 000g. Aspirate the supernatant from each 50-milliliter tube.Gently resuspend the beads and 0.5 milliliters of fresh HBSS. Transfer the combined beads with a P1000 micropipette to a new amber-colored glass vial, and store at four degrees Celsius. The filtration effect on a bead suspension comprising approximately 66%particulates is seen here.After filtration, the particulate content decreased to approximately 25%The adverse effects of filtration are the loss of beads and a slight loss in fluorescence intensity.