Name: mass cytometry: protocol for daily tuning and running cell samples on a cytof mass cytometer
Uploaded: 2012-11-02T14:00:00
Description: Watch this Scientific Journal Video about Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer at JoVE.com

我们想，感谢埃文·纽厄尔博士和博士肖恩Bendall反馈。我们还要感谢DVS科学博士和肖恩Bendall的样本的多含稀土的聚苯乙烯微球。我们感谢经费由美国国立卫生研究院授予U19 AI057229。

所有细胞为CyTOF样本必须固定和透化。这使得更大的含铱的DNA嵌入条目，并且还可以防止细胞裂解过程中的MilliQ水洗涤和再悬浮步骤之前立即注入流式细胞仪的质量。 1。启动的群众，流式细胞仪<ol><li>打开CyTOF的软件程序。选择仪器设置。在卡箱“选项卡，进入到右下角，然后单击”加热器“ON”按钮。打开加热器至少15分钟之前所需的时间，以便有足够的时间的雾化室地幔...

<ol>
	<li>Bandura, 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=Mass+cytometry:+Technique+for+real+time+single+cell+multitarget+immunoassay+based+on+inductively+coupled+plasma+Time-of-Flight+mass+spectrometry.">Mass cytometry: Technique for real time single cell multitarget immunoassay based on inductively coupled plasma Time-of-Flight mass spectrometry.</a> Anal. Chem. 81, 6813-6822 (2009).</li><li>Lou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-based+elemental+tags+for+sensitive+bioassays.">Polymer-based elemental tags for sensitive bioassays.</a> Angew. Chem., Int. Ed. 46, 6111-6114 (2007).</li><li>Majonis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curious+results+with+palladium-+and+platinum-carrying+polymers+in+mass+cytometry+bioassays+and+an+unexpected+application+as+a+dead+cell+stain.">Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.</a> Biomacromolecules. 12, 3997-4010 (2011).</li><li>Leipold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+mass+cytometry+methods+for+bacterial+discrimination.">Development of mass cytometry methods for bacterial discrimination.</a> Anal. Biochem. 419, 1-8 (2011).</li><li>Bendall, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+of+differential+immune+and+drug+responses+across+a+human+hematopoietic+continuum.">Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.</a> Science. 332, 687-696 (2011).</li><li>Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry+by+Time-of-Flight+shows+combinatorial+cytokine+expression+and+virus-specific+cell+niches+within+a+continuum+of+CD8++T+cell+phenotypes.">Cytometry by Time-of-Flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes.</a> Immunity. 36, 142-152 (2012).</li></ol>

在过去的几十年里，荧光流式细胞仪分析单个细胞的主力的方法，无论是在表面的表达和功能分析。然而，问题的荧光染料的光谱重叠的同步标记的数量有限制。虽然已有报道，实验中使用12个以上的同时标记所需的补偿量，使得此技术上有困难。 相反的荧光，质量仪率先由DVS科学使用螯合网站的聚合物的金属标签的抗体。1-3由于纯度的金属和ICP-MS质量分辨率，没有?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> CyTOF TM质量仪</td><td> DVS科学</td><td></td><td></td></tr><tr><td>洗涤液</td><td> DVS科学</td><td> 201071 </td><td>在水中的0.05％的氢氟酸</td></tr><tr><td>调优解决方案</td><td> DVS科学</td><td> 201072 </td><td> 0.5 ppm的镧，铯，铽，铥，铱，微量硝酸</td></tr><tr><td>含Eu的聚苯乙烯珠</td><td> DVS科学</td><td> 201073 </td><td>包含欧盟自然丰度同位素，珠子提供在约100万个/毫升</td></tr><tr><td>多含稀土的（La /公关/ TB / TM /卢） - 聚苯乙烯珠粒</td><td> DVS科学</td><td>尚未市售</td><td>包含上市镧系元素的自然丰度同位素</td></tr><tr><td>硝酸（浓） </td><td> Fisher Scientific则</td><td> A467-500 </td><td>越痕量金属的纯ICP-MS级</td></tr><tr><td>聚苯乙烯圆底管细胞滤网盖-5ML </td><td> BD </td><td> 352235 </td><td>用于过滤细胞样本，注射前</td></tr><tr><td>规范项目tuberkulin注射器1毫升</td><td>亨克萨斯狼</td><td> 4010-200V0 </td><td>无硅，无乳胶</td></tr><tr><td>规范项目注射器3毫升</td><td>亨克萨斯狼</td><td> 4010.000V0 </td><td>无硅，无乳胶</td></tr><tr><td> MilliQ水</td><td></td><td></td><td> 18MΩ纯水;必须不被存储在已用商业洗涤剂（由于其高的水平存在的钡）的玻璃或塑料瓶。 </td></tr><tr><td>企业所得税康闳</td><td> Sigma-Aldrich公司</td><td> Z273236 </td><td>酸性清洁剂</td></tr><tr><td>氩气</td><td>普莱克斯</td><td> AR 5.0UH-T </td><td>超高纯度99.999％ </td></tr></tbody></table>

mass cytometry: protocol for daily tuning and running cell samples on a cytof mass cytometer

在最近几年中，单个细胞的快速分析通常被执行采用流式细胞仪和荧光标记的抗体。但是，这个问题限制了数量的同时探针的荧光光谱重叠排放。相反，新的CyTOF质量流式细胞仪由DVS科学夫妇液体单细胞引入系统的ICP-MS 1而不是荧光，螯合聚合物含有高度富集的金属同位素被连接到抗体或其它特异性探针。2-5由于金属的纯度和质量分辨率的流式细胞仪的质量，有没有“光谱重叠”从相邻的同位素，因此，没有必要的补偿矩阵。此外，由于使用的镧系金属，有没有生物的背景，因此没有等效的自发荧光。有了一个质量窗口跨越原子质量103-203，理论上可达100个标签同时可以区分。目前，35个以上的信道可使用的螯合试剂可从DVS科学允许前所未有的夹层的免疫学的档案样本6-7 大规模流式细胞术的缺点包括每一个单独的金属同位素探针（没有相当于向前或侧向散射光），和事实，即它是一种破坏性技术（没有分拣恢复的可能性）的严格要求。流式细胞仪的质量的当前配置，还具有的小区传输率只有〜25％，因此需要较高的输入的细胞数。 日优化性能的质量仪需要几个步骤。优化的基本目标是最大化所需金属同位素（M）的所测量的信号强度，而形成的氧化物（M 16），将减少与任何所需的信号，在M 16的M信号强度和干扰最小化。第一步是热身机器，使一热，稳定的ICP血浆已经建立。其次，电流和补充气体流率的设置，必须在每日的基础上进行了优化。在样品的采集，最大细胞事件发生率是由检测器的效率和1000个细胞/秒的处理速度的限制。然而，根据较慢的细胞事件的发生率（300-500个细胞/秒）上的样品的质量，通常是可取的，以允许更好的分辨率细胞之间发生的事件，从而最大限度地提高对双峰和碎片完好汗衫。最后，足够的清洗的机器在一天结束时，可以尽量减少背景信号由于游离金属。

上述协议应做到四件事。首先，使足够的预热时间为流式细胞仪的质量会产生热，所需的最佳的信号和最小的氧化物形成的稳定的等离子体。二，流式细胞仪的质量，充分洗涤的MilliQ水和DVS的洗涤液（ 图9）将有助于减少吸附金属管和其他地区的机器的水平，有助于降低背景样品采集过程中（ 图8）。这也将有助于消除任何细胞，可能会卡在机器中，从而尽量减少残留样?...

Watch this Scientific Journal Video about Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer at JoVE.com

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

日常调整和优化的性能的CyTOF质量流式细胞仪所必需的步骤说明。上最佳的样品制备和流速的评论讨论

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