Name: use of the pyrimidine analog, 5-iodo-2&prime;-deoxyuridine (idu) with cell cycle markers to establish cell cycle phases in a mass cytometry platform
Uploaded: 2021-10-22T13:00:00
Description: Watch this Scientific Journal Video about Use of the Pyrimidine Analog, 5-Iodo-2Ã¢â‚¬Â²-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform at JoVE.com

作者要感谢Palak Sekhri，Hussam Alkhalaileh，Hsiaochi Chang和Justin Lyeberger的实验支持。这项工作得到了Pelotonia奖学金计划的支持。本材料中表达的任何意见、发现和结论都是作者的观点、发现和结论，并不一定反映 Pelotonia 奖学金计划的意见、发现和结论。

1. 制备 IdU 储备液
<ol><li>将5-碘-2'-脱氧尿苷（IdU）溶解在DMSO中至50mM的浓度。无菌过滤器，分装到10-50μL管中，并储存在-80°C</li>
	<li>从冰箱中取出 IdU，并在室温下解冻。在RPMI-1640中稀释IdU以制成终浓度为1mM的工作溶液。上下移液或涡旋混合。<ol><li>通常，将浓缩的IdU稀释到培养细胞的培养基（例如DMEM，IMDM等）中，或将其稀释到PBS中，以直接添加到外周血或骨髓抽吸物样品中。该预稀释步骤有助?...

<ol>
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J., Huo, Y., Chi, A., Kariv, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cryopreservation+on+delineation+of+immune+cell+subpopulations+in+tumor+specimens+as+determinated+by+multiparametric+single+cell+mass+cytometry+analysis.">Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.</a> BMC Immunology. 18 (1), 6 (2017).</li><li>Shabihkhani, 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=The+procurement,+storage,+and+quality+assurance+of+frozen+blood+and+tissue+biospecimens+in+pathology,+biorepository,+and+biobank+settings.">The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings.</a> Clinical Biochemistry. 47 (4-5), 258-266 (2014).</li></ol>

此处提供的示例演示了如何使用MCM平台分析细胞周期分布。还证明，细胞周期分析对时间和温度等实验条件很敏感，这是研究人员在考虑MCM进行细胞周期分析时必须考虑的重要考虑因素14。样品在短时间内储存，不超过一小时，其IdU掺入量将与其正常状态相当。样品在封闭系统中长时间（约2小时）将减少IdU掺入，但是，相对循环和非循环级分不会改变，允许粗细胞周期分析。低?...

质谱分析利用质谱的高分辨率和定量特性，可以检测大约40个参数。使用金属标记抗体代替荧光团偶联抗体，后者允许更多通道并产生最小的溢出1，2。与流式细胞术相比，MCM在细胞周期分析方面各有优缺点。MCM的一个主要优点是，大量的参数能够同时测量高度异质样品中大量免疫表型不同的T细胞类型的细胞周期状态。MCM已成功用于测量人骨髓3和端粒酶缺陷4的转基因鼠模型正常造血期间的细胞周期状态。对急性髓系白血病（AML）细胞周期状态的分析表明，细胞周期与对临床治疗的已知反应相关，提供了对功能特征的体内洞察，可以为治疗选择提供信息5。质谱细胞术细胞周期分析的第二个优点是能够测量可能与细胞周期状态相关的大量其他功能标志物。最近的工作已经能够通过使用IdU和针对BRU和rRNA6的金属标记抗体将蛋白质和RNA合成与细胞周期状态相关联。这种高度参数化的分析在连续分化过程中测量众多群体的细胞周期状态，使用目前的流式细胞术技术几乎是不可能的。MCM的主要缺点是缺乏与荧光流式细胞术中使用的DNA或RNA染色剂相当的DNA或RNA染色剂（例如DAPI，Hoechst，Pyronin Y等）。荧光染料可以相对精确地测量DNA和RNA含量，但这种精度只有在核苷酸碱基之间插层时这些染料的荧光特性发生变化时才有可能。因此，MCM分析无法以类似的精度测量DNA或RNA含量。相反，质谱细胞术细胞周期分析依赖于与细胞周期状态相关的蛋白质的测量，例如细胞周期蛋白 B1、磷酸化视网膜母细胞瘤蛋白 （pRb） 和磷酸化组蛋白 H3 （pHH3），并结合直接测量碘原子从 IdU 掺入 S 期细胞。这两种测量方法在正常细胞增殖期间产生高度相似的结果，但当细胞周期进程被破坏时可能会不一致。
测量每个细胞周期阶段的细胞数量对于了解正常的细胞周期发育以及细胞周期破坏非常重要，这在癌症和免疫疾病中很常见。MCM 使用金属标记抗体提供细胞外和细胞内因子的可靠测量;然而，S相的测量受到限制，因为基于铱的DNA插入器无法区分2N和4N DNA。为了定义细胞周期阶段，Behbehani开发了一种利用质量为127的IdU的方法，该方法落在质谱细胞仪的范围内，并允许直接测量S期3中的细胞。这种直接测量避免了对二抗或使用DNA变性剂（如酸或脱氧核糖核酸酶）的需求。结合细胞内循环标志物，它可以在实验模型中实现细胞周期分布的高分辨率。
该协议适用于MCM常见流式细胞术方案的细胞周期测量。我们的方法提供了一种方便和简单的方法来包含细胞周期参数。体外样品的IdU掺入仅需在37°C下孵育10至15分钟，这比大多数BrdU染色方案短，建议孵育时间为数小时3，7。IdU和BrdU掺入的样品可以使用蛋白质组学稳定剂固定，然后在-80°C冰箱中储存一段时间。这允许将大量 IdU 染色样品存档以进行批量分析，而不会降低样品质量。

<table>
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A3059</td>
			<td>Component of CSM</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-217-420</td>
			<td>Sample centrifugation</td>
		</tr>
		<tr>
			<td>Cleaved-PARP (D214)</td>
			<td>BD Biosciences</td>
			<td>F21-852</td>
			<td>Identification of apoptotic cells</td>
		</tr>
		<tr>
			<td>Cyclin B1</td>
			<td>BD Biosciences</td>
			<td>GNS-1</td>
			<td>G2 Resolution</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>Cryopreservative</td>
		</tr>
		<tr>
			<td>EQ Four Element Calibration Beads</td>
			<td>Fluidigm</td>
			<td>201078</td>
			<td>Internal metal standard for CyTOF performance</td>
		</tr>
		<tr>
			<td>FACS Tube w/ mesh strainer</td>
			<td>Corning</td>
			<td>08-771-23</td>
			<td>Cell strainer to remove clumps/debris before CyTOF run</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td>Cell culture growth supplement</td>
		</tr>
		<tr>
			<td>Helios</td>
			<td>Fluidigm</td>
			<td></td>
			<td>CyTOF System/Platform</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>Staining additive to prevent non-specific staining</td>
		</tr>
		<tr>
			<td>IdU (5-Iodo-2&#8242;-deoxyuridine)</td>
			<td>Sigma</td>
			<td>I7125</td>
			<td>Incorporates in S-phase</td>
		</tr>
		<tr>
			<td>Ki-67</td>
			<td>eBiosciences</td>
			<td>SolA15</td>
			<td>Confirmation of G0/G1</td>
		</tr>
		<tr>
			<td>MaxPar Multi Label Kit</td>
			<td>Fluidigm</td>
			<td>201300</td>
			<td>Metal labeling kit, attaches metals to antibodies</td>
		</tr>
		<tr>
			<td>Microplate Shaker</td>
			<td>Thermo Scientific</td>
			<td>88880023</td>
			<td>Mixing samples during staining</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>pentamethylcyclopentadienyl-Ir(III)-dipyridophenazine</td>
			<td>Fluidigm</td>
			<td>201192</td>
			<td>Cell identification during CyTOF acquisition</td>
		</tr>
		<tr>
			<td>p-H2AX (S139)</td>
			<td>Millipore</td>
			<td>JBW301</td>
			<td>Detection of DNA damage</td>
		</tr>
		<tr>
			<td>p-HH3 (S28)</td>
			<td>Biolegend</td>
			<td>HTA28</td>
			<td>M-phase Resolution</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Wash solution for cell culture and component of fixative solution</td>
		</tr>
		<tr>
			<td>p-Rb (S807/811)</td>
			<td>BD Biosciences</td>
			<td>J112906</td>
			<td>G0/G1 Resolution</td>
		</tr>
		<tr>
			<td>Proteomic Stabilizer</td>
			<td>SmartTube Inc</td>
			<td>PROT1</td>
			<td>Sample fixative</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td>Cell culture growth medium</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Acros Organics</td>
			<td>AC447810250</td>
			<td>Component of CSM/Antibody buffer, biocide</td>
		</tr>
	</tbody>
</table>

use of the pyrimidine analog, 5-iodo-2&prime;-deoxyuridine (idu) with cell cycle markers to establish cell cycle phases in a mass cytometry platform

细胞周期阶段的调节是细胞增殖和稳态的一个重要方面。控制细胞周期的调节机制的破坏是许多疾病的特征，包括癌症。对细胞周期的研究需要能够定义细胞周期进程的每个部分中的细胞数量，以及清楚地描绘每个细胞周期阶段之间的细胞数量。质谱细胞术（MCM）的出现为通过直接测量元素同位素进行高通量单细胞分析提供了巨大的潜力，并且通过MCM测量细胞周期状态的方法的开发进一步扩展了MCM的实用性。在这里，我们描述了一种在MCM系统中直接测量5-碘-2′-脱氧尿苷（IdU）的方法，类似于5-溴-2'-脱氧尿苷（BrdU）。使用这种基于 IdU 的 MCM 具有多种优势。首先，IdU在合成过程中迅速掺入DNA中，允许在短至10-15分钟的孵育中可靠地测量S期细胞。其次，IdU的测量不需要二抗或DNA降解。第三，IdU染色可以很容易地与细胞周期蛋白B1，磷酸化视网膜母细胞瘤蛋白（pRb）和磷酸化组蛋白H3（pHH3）的测量相结合，它们共同提供了五个细胞周期阶段的清晰描述。将这些细胞周期标记物与MCM可能具有的大量参数相结合，可以与许多其他指标相结合。

利用HL-60细胞和人骨髓抽吸物，可以显示实验条件如何影响细胞周期分布和分析。首先，必须建立门控策略以证明细胞周期阶段是如何得出的。在图1中，我们显示了单线态门的建立，这对于分离细胞碎片和双峰非常重要，从而建立单细胞群。对于细胞系，单线态门是进行细胞周期分析所需的全部（图2a）。对于人类样品，通常需要在细胞周期分析之前?...

Watch this Scientific Journal Video about Use of the Pyrimidine Analog, 5-Iodo-2Ã¢â‚¬Â²-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform at JoVE.com

Use of the Pyrimidine Analog, 5-Iodo-2ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â²-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

该协议调整细胞周期测量以用于质质细胞术平台。凭借质谱细胞术的多参数功能，直接测量碘掺入可以鉴定S期细胞，而细胞内循环标记物可以在一系列实验条件下表征每个细胞周期状态。

使用嘧啶类似物5-碘-2′-脱氧尿苷（IdU）和细胞周期标志物在质谱细胞术平台中建立细胞周期期

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the ...

该协议使我们能够使用质谱细胞仪在单细胞水平上检查细胞周期，这使得可以进行几项新的研究和临床研究。这种技术的主要优点是它的简单性。IdU直接添加到细胞中，无需进一步处理或抗体。我们已经使用这种技术来研究干细胞和祖细胞区室内急性髓系白血病患者的细胞周期状态。这表明这些特定细胞类型的细胞周期特性可能与这些疾病患者的临床结果相关。该技术可用于了解稀有癌细胞的生长特性和了解免疫细胞亚群的增殖。在将IdU添加到细胞样品之前和之后，将细胞保持在感兴趣的生长条件下。在这种情况下，在5%CO 2的37摄氏度加湿培养箱中用IdU标记细胞，将浓缩的IdU储备溶液稀释到细胞的生长培养基中。然后将样品转移到生物安全罩中，并向每1毫升细胞中加入10微升一毫摩尔IdU。将细胞放回培养箱中10至15分钟，然后将样品转移到单独的锥形管中。通过在15毫升锥形管中离心收集细胞，并将沉淀重悬于每管200微升PBS中。如果需要，用适当的活/死染色标记细胞以标记死细胞。活/死染色后，用CSM洗涤的培养基淬灭细胞，然后用25微升16%多聚甲醛固定细胞。在室温下孵育10分钟后，通过离心收集细胞。加入5至15毫升CSM，再次离心。将沉淀重悬于补充有10%二甲基亚砜的500微升细胞染色培养基中。然后将细胞转移到较小的管中，将它们储存在负80摄氏度下。要标准化其细胞周期分析，请将文件导入适当的流式细胞术分析软件程序，并创建事件长度与191铱的双轴图。这些细胞将形成一个独特的明亮铱高群体。更改事件长度刻度以使单元格显示得更突出。然后创建一个单重门以排除 50% 到 60% 的双峰和碎片，并设置残余和高斯偏移参数以去除任何剩余的双峰和碎片。要设置 S 相门，请创建 IdU 与增殖标记的双轴图。S期IdU阳性细胞将形成一个独特的群体。对于 G0/G1 和 G2/M 门控，在 IdU 与细胞周期蛋白 B1 图上建立门。G0/G1 期将为细胞周期蛋白 B1 低，IdU 阴性，G2/M 期将为细胞周期蛋白 B1 高，IdU 阴性。要针对特定细胞类型进行门控，请仅在细胞周期蛋白 B1 与 IdU 图上绘制 S 期细胞，以帮助确定 G0/G1 期和 G2/M 期细胞之间的分离。在细胞周期蛋白B1高种群周围画一个门。在某些情况下，这可能很难辨别，但细胞周期蛋白B1的水平约为S相群体的前5%，通常是G0/G1和G2/M相栅极之间的断点。驻留在前一个门内的细胞将是G2 / M相群体，而其余的将是G0 / G1相群体。要建立 G0 期群体，请创建磷酸化视网膜母细胞瘤蛋白与 IdU 图。G0期将由磷酸化的视网膜母细胞瘤蛋白低，IdU阴性群体表示。由于活跃的循环群体将表现出高磷酸化的视网膜母细胞瘤蛋白和IdU掺入，因此G0相门可以在此边界上绘制，因为它通常在两个不同的群体中表达。如果 G0 门难以定义，请将 S 期群体设置为活跃群体，并绘制包含前 90% 至 100% 磷酸化视网膜母细胞瘤蛋白高群体的门。门外的磷酸化视网膜母细胞瘤蛋白低群体通常是G0群体，而落在该门中的pRb水平的G0 / G1细胞是G1细胞。对于 M 期门控，通过轴向图创建 IdU 与磷酸化组蛋白 H3。M相将作为磷酸化组蛋白H3群体中非常小的细胞部分被观察到。G2/M 群体中 pHH3 水平较低的细胞是 G2 细胞。如果 IdU 掺入失败或不可能，请使用 Ki67 和磷酸化视网膜母细胞瘤蛋白定义循环和非循环细胞组分。双正总体将表示与 G1、S、G2 和 M 阶段相关的活跃循环总体。双低人口将表示与 G0 阶段相关的非循环人口。建立所有门后，从门导出数值。每个周期中的百分比可以通过从组合群体中减去单个群体来实现，以生成每个单独细胞周期阶段的数值，以便随后进行图形和统计分析。单线态门的建立对于分离细胞碎片和双峰以允许分离单细胞群非常重要。然后可以对单个细胞进行细胞周期分析。IdU标记和下游细胞周期分析会受到时间和温度的显着影响。例如，在封闭容器中或在位置之间运输中停留时间过长的细胞将具有减少的S相分数，这对于细胞周期分析将不准确。细胞样品保持一小时或更短的时间仍然显示出正常的细胞周期分布，表明快速运输可能不会对分析产生负面影响。冷冻保存的细胞可能需要很长的平衡期才能使细胞恢复到活跃的细胞周期，这可能仍然不能反映冷冻保存前的细胞周期状态。不同的细胞类型也可能受到维持条件的差异影响。例如，在这个人类骨髓样本中，与来自同一样本的单母细胞相比，T细胞显示出细胞数量的减少。质谱细胞术的另一个好处是能够区分细胞周期停滞或细胞周期分布异常的细胞。该技术的一个主要优点是它很容易与其他质谱细胞术测量兼容，例如表面表型，染色质结构，细胞内细胞因子刺激，这些都可以与细胞周期状态相关。我们已经使用这种技术来研究免疫细胞的衰竭，衰老，并表明急性白血病患者对化疗的反应可能与白血病细胞的细胞周期特性相关。

Research

JoVE Journal

Genetics

Use of the Pyrimidine Analog, 5-Iodo-2&prime;-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...