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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(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。

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

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

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  

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

科研

化学

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

利用停流荧光和标记的核苷酸来分析驱动蛋白的ATP周转周期

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

羰基饰卡宾的白色磷的活化制备方法和用途

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

反应执行使用连续流处理的实时监控:3  - 乙酰基香豆素的制备为例

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment allows for testing of various proposed ocean / hydrothermal fluid chemistries that could have driven reactions toward the origin of life in environments on the early Earth, early Mars, or even other worlds such as the icy moons of the outer planets. We show experiments that include growth of chemical garden structures under anoxic conditions simulating the early Earth, inclusion of trace components of phosphates / organics in the injection solution to incorporate them into the structure, a switch of the injection solution to introduce a secondary precipitating anion, and the measurement of membrane potentials generated by chemical gardens. Using this method, self-assembling chemical garden structures were formed that mimic the natural chimneys precipitated at submarine hydrothermal springs, and these precipitates can be used successfully as flow-through reactors by feeding through multiple successive &#8220;hydrothermal&#8221; injections.

We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents.

化工园为直通式反应器模拟自然热液系统

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment ...

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

的凹孔隙PDMS珠微泡制作

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

柱后衍生用反应流高效液相色谱柱

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

电化学阻抗谱作为电化学速率常数估计的一种工具

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...