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的达到200°C。 </li><li>更换白色规范项目3毫升注射器充满MilliQ水的一个新的注射器注射泵。 </li><li>清洗喷雾器。反冲通过使用注射器和油管拉5％citranox，两个较小的液体导入口和较大的气体导入口的2-3倍，通过雾化器。重复反冲用MilliQ水tØ去除残留的citranox。 </li><li>检查确认前的CyTOF面板上的灯被点亮，如在图1中。 </li></ol><img alt="图1" src="/files/ftp_upload/4398/4398fig1.jpg" /> 图1。CyTOF面板灯之前启动。 <ol start="5"><li>打开氩气罐的阀门。这应该引起“氩”光的前面打开。 </li><li>将雾化器，使气体入口。拧下使气体管道连接器，黑色橡胶O型圈，白色锥形塑料片拆下。注意更广泛的“转向节”雾化器上的气体导入口干。放置在连接器上的气体导入口。黑色的O形圈放置在进气口端部的，和过去的较宽部分的端口干强制的O形环。将顶部的窄端的白色圆锥形塑料片上指出，和螺杆化妆气体管道上。 </li><li将雾化器的液体引入管。小心雾化器的插入小管的开口。将有两站路，觉得站上的微量移液器相似。首先，按，直到你遇到的耐光性的小管应在玻璃的直线段的结束。其次，用力按下强制结束的玻璃直线段进一步进入装修，并拧紧螺丝固定密封。雾化器的一端插入到在加热套的白色开口。 </li><li> RFG控制器“选项卡的”仪器设置“窗口中，按下”开始“等离子”。由于雾化器已经插入，然后按“OK”。软件会打开冷水机组，启动气体的流量，并点燃了等离子。该探测器电压会上升。将有一个弹出式窗口，说：“等离子的启动顺序已成功完成”完成后，按“OK”。新的灯应该是在前面板上（ 图2 </strong<html> &gt;）。 </li></ol><img alt="图2" src="/files/ftp_upload/4398/4398fig2.jpg" /> 图2。CyTOF面板灯等离子点燃后，启动成功完成。 <ol start="9"><li>注射器泵的开启。在右上角，按绿色的“播放”按钮开始注射泵，并开始流动的液体通过管道和雾化器喷射而出。默认的注射器设置为3毫升水。为了样品流经样品定量环，注射泵必须定期更换的，当它运行的水。在屏幕的顶部，是一个计数器，其指示多少液体流出的注射器泵。当此计数器达到“3.000”或柱塞击中的注射器的端部时，将停止的注射器泵。当重新填充注射器，你必须打“停止”，而不是蓝色的“暂停”按钮，重设计数器。 </li></ol>E“&gt; 2。每日的群众流式细胞仪的校准<ol><li>流式细胞仪的质量大约需要20分钟，以达到校准前的一个热点，稳定的等离子体。的调整步骤的目的是最大化，同时保持所需的信号Tm169或Tb159“Gd155”（氧化物La139 + O16）信号在3％以下价值较高。点击获取设置“按钮，打开数据采集设置”窗口中。在分析物“选项卡上，单击”周期表打开的同位素选择面板。选择DVS科学调优解决方案手册中列出的同位素，然后单击“保存”。 </li><li>点击阅读按钮，每次读取窗口打开大容量（ES）的同位素每。进入一个新的号码，然后按“ENTER”选择“脉冲计数”，重新调整y轴。 </li><li>填写一个绿1毫升注射器与规范项目调整解决方案。打开的的样品端口选择“LOAD”，注入450μl的调优解决方案，然后旋转选择器“注入”。在质量校正“选项卡上的数据Acquisit中<html>离子设置“窗口，按”运行“监控群众都流淌着。窗口的左侧是较低的质量，而右侧是较高的质量。注意：将已经在TOF 9400-9800区域的信号，由于氩气中的（ 图3）的氙同位素。等待，直到调优解决方案同位素开始出现条纹，这显示（ 图4）。 </li></ol><img alt="图3" src="/files/ftp_upload/4398/4398fig3.jpg" /> 图3。质量校正“选项卡窗口，显示各种同位素的背景水平后，洗涤液和清水冲洗。 9400-9800 TOF周围区域对应的氙同位素氩气中的，总应该存在。 <a href="http://www.jove.com/files/ftp_upload/4398/4398fig3large.jpg" target="_blank">点击此处查看大图</a> 。 “SRC =”/ files/ftp_upload/4398/4398fig4.jpg“/&gt; 图4。质量校正“选项卡窗口，显示垂直条纹各种同位素DVS调优解决方案。 <a href="http://www.jove.com/files/ftp_upload/4398/4398fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <ol start="4"><li>返回的质量（）每次读取的窗口，按下绿色的“播放”按钮，在左上角。在步骤2.1中选择的每个同位素是由不同颜色的线表示。默认的X轴是“时间”，以秒为单位。监视水平的不同的同位素，直到他们是稳定的。 </li><li>调谐电流。在“参数”选项卡的数据采集设置“窗口中，从下拉菜单中选择”当前“。让我们开始= 0，完成= 10，步长值= 0.5，沉淀时间= 200，然后按“保存”。点击返回每次读取的窗口和打比赛的质量（ES）。的x-轴的现在是“当前”。记下的信号的同位素峰（ 图5），和r<html> ECORD。在DAC通道设置“选项卡的”仪器设置“窗口中，向下滚动到”当前“，然后输入这个新的价值。按“实际电流值”，然后单击“保存”。 </li></ol><img alt="图5" src="/files/ftp_upload/4398/4398fig5.jpg" /> 图5。电流调节配置文件。 <ol start="6"><li>调整的化妆气体。更改下拉菜单中的“化妆气。”让我们开始= 0.55，= 0.95，步长值= 0.05，稳定时间= 200结束。按保存。每次读取的窗口和打比赛的质量（ES）。 x轴是现在“化妆气体”的流量。记录下的信号的同位素峰。观察的“Gd155”附近的端部的信号（ 图6）的快速增加。返回的DAC通道设置“选项卡，向下滚动到”化妆气“，进入这个新的价值。按“实际电流值”，然后单击“保存”。 </li></ol>IGURE 6“SRC =”/ files/ftp_upload/4398/4398fig6.jpg“/&gt; 图6。化妆气体调节配置文件。 <ol start="7"><li>记录率所需的同位素比氧化物。第二个450微升注射调优解决方案。在“参数”选项卡上，从下拉菜单中选择“时间”，让我们开始= 0，最终解决时间= 200 = 10，步长值= 1，。按“保存”。每次读取的窗口，按下“播放”质量​​（ES）。行后已稳定（ 图7），使用光标阅读的价值在左上角方块（数值越高，Tm169或Tb159）和“Gd155”价值较高的值（&lt;3％）。这些值也被保存在一个调谐文件以供将来参考。 </li></ol><img alt="图7" src="/files/ftp_upload/4398/4398fig7.jpg" /> 图7。优化后的电流和化妆的气调优解决方案的信号强度测量。 <ol start="8"><li>清洗样品PLE循环。注入3毫升的MilliQ水通过进样环DVS的洗涤液，随后加入500μl。监控进度的清洗，按“运行”，在“质量校准”选项卡。洗净直到条纹的强度（ 图4）开始减小，然后按照由3毫升的MilliQ水和显示器，直到下降到背景水平（ 图3）。 </li></ol> 3。运行样品<ol><li>打开采集窗口。默认设置：做[分析=-THE-FLY，信号分析=双，双计校准数据。降噪选择。如果需要的话，可以改变任何这些。输入文件名。 必须将所有文件保存到E :/ /驱动器。 </li><li>设置采集的延误。注册细胞的的样品循环和雾化器之间的管道的长度约30秒的事件，由于会有轻微的延迟。因此，“采集延迟”的总和和“探测器第一能力延迟“应该是至少30秒。探测器稳定性延迟应该是至少20秒。 </li><li>设置收集数据的时间。 “采集时间”是你要运行您的样品的秒数。填充整个450微升样品环，将需要600秒才能完成。建议，50-100秒被添加到运行时间的结束，以尽量减少样品结转。 </li><li>再悬浮的样品。为了尽量减少积聚在本机中的盐，它被认为至少两个做在MilliQ水中洗涤结束时的细胞染色，然后由一个最后的再悬浮在MilliQ水中。 MilliQ水的体积用重悬细胞沉淀将取决于您开始使用的细胞和细胞后恢复整个染色过程的数目而有所不同。一个很好的起点为10 6细胞/ ml（启动）。再悬浮后，通过细胞过滤器过滤，以除去聚集。 </li><li>启动示范运行。当前样本输入新的文件名。<html>样品循环阀切换到“LOAD”，注入样品，切换到“插入”，然后点击“运行”，在采集窗口。 </li><li>注册细胞的活动。将出席在细胞水平的标记同位素/标记在每个垂直通道（ 图8）集合的快照窗口。流式细胞仪的质量可以注册1000个细胞/秒，细胞活动之间的更好的分辨率通常在300〜500个细胞/秒。这相当于平均〜1细胞/屏幕刷新。它可能需要长达30秒前的细胞事件计数器开始攀升。这是由于“飞”数据处理;失去任何细胞事件信息。 </li></ol><img alt="图8" src="/files/ftp_upload/4398/4398fig8.jpg" /> 图8在样品采集窗口运行，示出了三个单独的细胞事件关联的元素相对应的斑点的水平行。ww.jove.com/files/ftp_upload/4398/4398fig8large.jpg“目标=”_blank“&gt;点击这里查看大图。 <ol start="7"><li>细胞活动的质量。请注意，任何的背景裸奔在每个同位素通道，以及细胞事件发生率。流式细胞仪的质量承认“电池事件”在上述背景下的信号有一定的增加，使游离的金属或者未结合的抗体可以改变的“电池事件”计数的背景信号。此外，运行在过高的浓度的细胞可能会导致双峰的事件的数目的增加。背景可以减少额外的清洗或更大的细胞稀释。双峰更大的细胞稀释液，在运行时增加的成本可以最小化。 </li><li>完成采样数据采集。当采集时间已经达到，数据采集完成。会有点延迟，前一个弹出式窗口的“确定”按钮;再次，这是由于“飞行”数据处理。样品采集也可以停止普利马特urely通过点击“停止”按钮。在这种情况下，所有的细胞已经注册的事件将被计算和处理到输出文件中。 </li><li>至少500μL的MilliQ水之间每个样品注射也有助于最大限度地减少样品交叉污染。 </li></ol> 4。使用后清洗机<ol><li>洗净的解决方案。在最后的运行样品和样品循环用水冲洗，洗涤液注入450μL的样品环。监控释放游离的金属中的的质量校准“选项卡的数据采集设置”窗口，在步骤2.8（ 图9）。当游离金属信号开始下降，用3毫升的MilliQ水冲洗和监测，直到达到背景水平。 </li></ol><img alt="图9" src="/files/ftp_upload/4398/4398fig9.jpg" /> 图9。质量校正“选项卡窗口，在运行后DVS洗涤液清洗。垂直条纹TOF地区9800-11000抗体标记同位素被清理出来的机器。 TOF 11500左右的垂直条纹对应的两个红外嵌入同位素。 <a href="http://www.jove.com/files/ftp_upload/4398/4398fig9large.jpg" target="_blank">点击此处查看大图</a> 。 5。关闭机器<ol><li>在的RFG控制器“选项卡”仪器设置“窗口中，选择”停止等离子“。当程序完成后，将有一个弹出式窗口，询问您删除雾化器。按“OK”。在卡箱“选项卡上，关闭加热器。关闭阀门的氩气供应。 </li><li>去除雾化器。将雾化器雾化室，仔细地拉向后。从步骤1.6拧下的样品引入管和放置一旁。拧开化妆的气体导入连接稍微放松，然后将雾化器。离开接头拧在了一起，将减少的机会升osing的黑色橡胶O型圈和白色锥形塑料片。 </li><li>清洗喷雾器。使用注射器和油管在步骤1.3中，用5％citranox反冲的雾化器的两个端口。商店，覆盖直到下次使用在5％citranox的。 </li></ol>

<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>

作者在人体免疫监测中心，服务中心在斯坦福大学收取使用费，仅收回成本的分析，包括质量仪的工作。

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

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

mass-cytometry-protocol-for-daily-tuning-running-cell-samples-on

Research

JoVE Journal

Bioengineering

22.7K 次观看.  Stanford University. 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

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

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

科研

生物工程

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

一个手机上的宽视场荧光显微镜和荧光成像流式细胞仪

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案：二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

基于海胆大白鲨A接地采样：仿生为设计一个协议

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

全身质谱成像红外基质辅助激光解吸电喷雾电离（IR-MALDESI）

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

使用CRISPR并发剂的小鼠小肠组织中多基因敲除的方案

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Methods Article

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪