作者特别感谢以下人士：布鲁斯·巴格韦尔他们的技术和知识贡献，经过多年的发展，这些方法（Verity的软件公司），NadègeBercovici（IDM），Lizanne布雷斯林（Zynaxis细胞科学和PTI研究的） ，布莱恩·格雷（PTI研究），月费雪（达特茅斯医学院），爱丽丝吉万（达特茅斯医学院），贝齐·奥尔森威廉（SciGro公司），和玛丽·沃（达特茅斯医学院）。他们也想感谢鲍登学院2006级年度课程研究方法与应用流式细胞仪检测，其产生的数据， 如图2所示。 流式细胞术在Roswell Park癌症研究所的流式细胞仪检测实验室，成立于设备补助部分由美国国立卫生研究院的共享仪器程序，并接收从Core资助（5 P30 CA016056-29），从国家的支持Roswell Park癌症研究所癌症研究所，并在Abramson癌症中心的流式细胞仪和细胞分选资源宾夕法尼亚大学的实验室，成立于部分由美国国立卫生研究院的共享仪器程序的设备补助，并接收由美国国立卫生研究院的支持＃2P30 CA016520从美国国家癌症研究所。在图3和图5所示的工作也得到了支持部分由SBIR授予EB00228从国家生物医学成像和生物工程研究院（NIBIB）颁发给，PTI研究公司

1。一般的薄膜标签用PKH26细胞示踪染料（编号25，图1） <ol><li>使用无菌技术的步骤1.1 - 1.9。准备〜10 7人末梢血单核细胞或淋巴细胞（人外周血单个核细胞，hPBL），此外，最后的300 xg离心自旋使用实验室的标准方法，以尽量减少血小板污染。重悬细胞于10 7个/ ml，在HBSS 1％BSA中，置于冰上，保留500μl的等分试样（5×10 6个细胞），在步骤2中使用。 </li><li>地点5×10 6个细胞（500微升）在12×75毫米同轴的聚丙烯管中。用3.5毫升HBSS洗涤一次。小心吸出上清液，留下残留的液体不超过15-25微升，但小心不要删除单元。使用此管准备2倍的细胞悬浮液的步骤1.4。 </li><li>在细胞洗涤在步骤1.2中，加入0.5ml的稀释液的Ç标签车辆（从PKH26GL套件）一个12 x75毫米的圆锥形的聚丙烯管中。使用此管的准备2倍的PKH26解决方案，步骤1.5。 </li><li>洗涤后的细胞沉淀中加入0.5毫升稀释液的的Ç标签车辆从步骤1.2和吸液和分配的3-4倍，得到单细胞悬浮液（2×细胞）。避免泡沫的形成和过度的混合，这可能会降低细胞活力和恢复。 </li><li>后，立即在步骤1.4中制备的2倍的细胞悬浮液中，准备了2倍（4μM）染料溶液中加入2.0微升1.0mM的PKH26染料库存在乙醇（从PKH26GL套件）的稀释液的制备在步骤1.3和旋涡的C管均匀分散。 </li><li>步骤1.5后，立即准备2倍的染料溶液，迅速 ​​移液管的2倍细胞悬浮液从1.4到2倍的染料溶液步骤，并同时吸液和分配的3-4倍，完全分散细胞的染料。 不要加入1.0毫米染料直接细胞;倒到2x染料的2个细胞，或增加2个细胞的2倍，而涡旋式染料。由于染色几乎是即时的，这种方法产生统一强度比推荐的方法（ 图2）。 </li><li> 1分钟后，加入1.0毫升的热灭活血清或HBSS 5％的BSA停止染料吸收到细胞膜。如果使用足够的蛋白质，形成染料聚集体，颗粒细胞在洗涤步骤，导致意想不到的标签与其他细胞的实验中存在的风险。如果与10％热灭活血清（CM）或HBSS 1％BSA的介质是被用来作为停止的试剂，在15毫升锥形聚丙烯管中进行染色，并添加至少为5.0毫升停止试剂，以确保所有的吸附未掺入的染料。 </li><li>标记的细胞离心5分钟@〜400×g下。小心吸出上清液，而不删除单元。洗涤沉淀两次用4毫升CM或HBSS 1％BSA的，分散粒料之前recentrifugation的。为了尽量减少残留的染料吸附在管壁上，最大限度地提高洗涤效率，转移到一个新的聚丙烯管细胞后，网络连接第一个悬浮。注：充分染的细胞会表现出不同的颗粒粉红色的色调。 </li><li>洗过的细胞沉淀重悬在1.0毫升的HBSS 1％BSA。计数细胞，测定细胞恢复，以及调节音量，得到的最终浓度为10 7个/ ml。通过仔细的愿望，电池回收应≥85％。如果细胞恢复&lt;70％时，应查明原因，然后再继续。提款150微升等分试样（1.5×10 6个细胞）和在步骤2中使用的在冰上地方。 </li></ol> 2。制备仪器和实验设置控制（ 表1） <ol><li>等分试样50微升（5×10 5），从保存在步骤1.1的细胞悬浮液到每个五1.8 ​​毫升Eppendorf管中：1，3，4，5，和7的未染色细胞。等分试样50微升（5×10 5），的PKH26 位置细胞到每个三级管从步骤1.9：2，6和8。 </li><li>管1-8，加入10μl的IgG嵌段（100微克/管的IgG;见表试剂）并在环境温度（20-25℃）孵育10分钟。 </li><li>加入饱和量的抗体（们）表示于表1中的管4，5，7，和8，并孵育30分钟，在室温和避光的所有样本（管1-8）。 </li><li>加入1.5毫升的HBSS 1％BSA的所有样品，沉淀细胞通过离心分离（5分钟@ 400 xg离心），并用1.5毫升1％BSA的HBSS洗一次，用小心抽吸，以避免细胞的损失。 </li><li>每个样品重悬在500微升的HBSS 1％BSA的。您的流式细胞仪上进行分析的样品，如果需要转移到一个12×75毫米的圆形底部管。管3，第6，第7和8中加入10μl的100微克/毫升7-AAD每日工作库存（见表试剂），如表1中所示。在冰上孵育30分钟，在使用前，在流式细胞仪安装和染色验证（步骤3）。 </li></ol> 3。流式细胞仪安装和染色验证<ol><li>验证流量cytome后运行正常，使用实验室的日常质量控制程序。验证信号可以很容易地被检测到在要使用的每个光谱窗口，和检测器响应是线性正比于在窗口中的信号强度，用于增殖监测14。 </li><li>收购管1 FSC与SSC数据使用线性显示比例。调整每个检测器的放大的淋巴细胞群，使得落在点图的左下象限，是不是断开规模两个参数中的任何一个中，并且不被中断，由于阈值化。收集所有的样品在步骤3.3 - 3.7非门FSC与SSC数据。 </li><li>采集数据管1，不使用色彩补偿所有4个荧光检测器和对数显示比例。调整各检测器的高电压（HV）放置规模少/无细胞积聚在第一信道的未染色的淋巴细胞的自发荧光。设置每个直方图的分析边界对应于最亮的2％的未染色细胞。 </li><li>不使用色彩补偿和HV设置在3.2和3.3，采集数据，收集管2 FSC，SSC，在所有4个荧光检测器的信号。 PKH26荧光用于监视检测，确认所有的的PKH26 POS细胞出现规模作为一个单一的对称峰在第三 -4 个十年，数/细胞中的最后一个通道。如果有多个峰，或峰形歪斜，重复步骤1，，与盐小心注意尽量减少和混合技术（ 图2）。如果有必要，调整染料的浓度。 </li><li>使用步骤3.3的设置，采集数据，收集管3 FSC，SSC，在所有4个荧光检测器的信号。用于监视7-AAD荧光检测，确认7-AAD的POS细胞下降2％以上步骤3.3（ 即非活细胞得到很好的解决边界建立可行的7-AAD 阴性细胞）。 </li><li>使用步骤3.3的设置，采集数据，收集管4 FSC，SSC，在所有4个荧光检测器的信号。检查未染色的细胞（ 即 ，秋天的FITC检测步骤3.3建立高于2％的边界），CD8的POS细胞得到很好的解决。管5重复和验证未染色细胞（ 即下跌超过2％的边界成立于APC检波器的步骤3.3），CD8的POS细胞得到很好的解决。 </li><li>使用步骤3.3中设置，采集数据，收集管6，第7和第8 FSC，SSC，在所有4个荧光检测器的信号。 </li><li>使用收集管1-5和色彩补偿软件，建立一个重叠矩阵，每个荧光探测器被用来监视其他三个荧光染料的颜色列表模式下的文件。应用此矩阵样品6到列表模式文件，并确认存在PKH26实验室鹅岭不改变的能力来检测7-AAD 位置细胞。 </li><li>应用颜色重叠矩阵，列表模式下的文件，样品7和第3.8步验证是：a）很好地解决亚群（CD3 负的 CD4 负 ，的CD3 POS CD4 neg和的CD3 位置 CD4 POS）可以识别的FITC与APC散点图，和b）的存在下，抗-CD3-FITC和抗-CD4-APC不改变7-AAD 位置细胞的能力来检测。如果存在下，抗-CD3-FITC改变PKH26检测器上收集的数据的高2％的边界，调整所要求的边界。 </li><li>应用的颜色重叠矩阵，从步骤3.8样品8到列表模式文件。如果存在的PKH26标记改变那些使用自体荧光的步骤控制在3.3 FITC 7-AAD或APC探测器的2％的边界，调整边界（IES）需要使用管7 表1，并确认其仍然是能够disting的uish CD3 位置 CD4 POS机 ，的CD3 POS CD4 负 ，和CD3 负的 CD4 阴性细胞使用调整后的边界（IES）。 </li></ol> 4。细胞分裂的监测染料稀释扩散模型选择<ol><li>确定代与代之间的间距，这是依赖于在您的流式细胞仪的数目的信道和对数十年。对于数字仪表，确定这个值，通常为4或5几十年来，在数字信号处理器的数目的箱柜。在模拟仪器，数十年来很少是整数，精确的建模需要的准确数字几十年来用实验方法确定。要做到这一点，从校准与制造商分配的相对强度的荧光珠的混合物获取的数据在相同的检测器在实验中使用的高电压设定。胎圈的峰的位置在int的方面允许的对数刻度的校准每个日志十年的ensity范围。具体而言，这是通过绘制每个胎圈类型对日志的制造商指定的值的信道数目。胎圈的数据值的最佳拟合直线的斜率给出了每个通道的相对强度单位的数目。乘以的信道数，然后将这个值是日志数十年的满刻度，从其中可以计算出到2倍的强度降低的现象（ 即 ，女儿代间距）相对应的数量的信道14的数目。 </li><li>决定是否使用一个固定的几代人之间的间距，或允许浮动的间距。 A 标准 （固定）设置将使用在步骤4.5确定指定的位置，每一代代间距值时通常使用的直方图没有明显的峰值。一个浮动设置允许的直方图的形状来确定每个代峰值位置时，通常使用distinguishablË代峰是显而易见的。 </li><li>决定是否使用一个固定的峰宽各代或浮动宽度。一个固定的宽度使用SD未刺激的对照样品各代建模和计算时，通常选择的样本缺乏区分的代峰。一个浮动的宽度允许程序独立地改变SD每一代都最适合用于区分的代峰。 </li><li>执行程序中的扩散分析模块（ModFit LT版本3.3）。刺激PKH26 位置设置为进行分析（ 例如 ，刺激96小时培养PKH26 位置细胞复染按照在表1中管8）的数据文件从装入。 </li><li>选择的参数进行分析，在这种情况下PKH26（585/42）上可行的门控（7-AAD 负。）CD3的POS淋巴细胞和FSC与SSC排除小碎片和大团聚体（ 图3）。在定义这些地区要小心爆炸通常包括前向散射面积，并注意CD3的表达可能是下调制刺激的培养。 </li><li>使用扩散向导创建一个新的扩散模型。使用打开数据文件 （“开始”选项卡上）中，装入未刺激PKH26的POS控制文件和定义的位置高峰在父母的信道分配相应不可分割的细胞。 </li><li> 分析该文件为刺激时PKH26 位置控制，注意到父母峰的位置和宽度（标准差）的值。如果需要一个固定的峰宽（SD），检查锁定SD。 </li><li>负载：PKH26 负控制（ 例如 ，PKH26 阴性细胞培养96小时的复染的管7 表1）。调整世代数 ​​的通过设置峰值通道的最暗的代以上的PKH26 负控制。这就决定了NUMBER的女儿代模型可以准确地适应，通常是6-9代。 </li><li> 打开刺激的样本（ 例如 ，刺激96小时培养PKH26 位置细胞复染管8在表1中）的数据文件，并确认父母的峰值位置和SD，如在步骤4.7中定义的区域不变。如果需要固定的世代间隔，选择“ 标准”模式的选项，否则选择浮动选项。 </li><li> 分析每一个实验性的文件中的数据集，在步骤4.9中定义的使用相同的模型。父母的峰值位置的一些小的调整可能是必要的视觉和减少的卡方值（RCS）最适合。 </li><li>记录所需的增殖的最佳拟合数据集合中的每一个实验性的文件产生的度量。可能指标的全面描述见参考文献。 22。 </li></ol>

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·汉弗莱，JD Tario，Jr。和PK华莱士已经收到了预商用的细胞跟踪试剂从Life Technologies，Inc。和BD Biosciences公司进行评估。 AD Bantly和JS Moore已收到预商用的细胞追踪试剂的评价从Life Technologies，Inc。和资金从PTI研究，​​公司为预商用表征的各种CellVue细胞示踪染料。 K.缪尔黑德是受雇于公司，提供咨询服务Phanos科技有限公司（所有者的PKH和CellVue染料），并提供Sigma-Aldrich公司和分子靶向技术，备份技术支持（SciGro，这些分销商染料）。 生产和免费使用这篇文章是由Sigma-Aldrich公司。

这里介绍的方法是在我们的联合实验室hPBMC标签用膜染料13,16,18的和T淋巴细胞亚群的表型和增殖跟踪，无论是膜或蛋白染料2,11,13,16，使用最可靠提供最佳的结果， 18。正如图1和图2中示出，明亮的统一的标签是最容易实现的生理盐的存在下，通过限制使用的混合技术，快速，均匀的曝光的所有单元格，以相同浓度的染料中的结果。因为划分的脂质双层膜染料染色时，其他变量，改变染料浓度，还可以影响标签的效率。例如，标签圆底聚苯乙烯管效率较低的盐冲洗稀释Ç的悬浮之前也减少由于染料的吸附在管壁上，特别是游离的染料浓度在染料浓度较低。这两个因素往往会得到更广泛的染色分布时相比，标记操作可以使用圆锥形底部的聚丙烯管中（ 图3，图4和 结果未显示）。样本的年龄和类型也可以影响峰宽，甚至被用来优化的染色过程。例如为的PKH26 位置淋巴细胞分离出新鲜抽血，范围从14-20％（参考图3，13日和未发表的结果），而24小时老血液样本或TRIMA的pheresis过滤器范围内分离出淋巴细胞的简历，个人简历25-30 ％（ 图4和参考文献18）。 染色的均匀性和非活细胞可以从分析中排除既影响染料稀释曲线，这反过来又影响的扩散模型的选择，以适应所观察到的数据（ 图5和图6是显而易见，是否区分的女儿峰的程度</strong&gt;）。这里使用的是，虽然ModFit（真理图软件House，托普瑟姆的。，ME）的软件用于生成等指标的扩散指数和前体频率（ 图3，图5和图6，表3）作为一个例子，其他软件包还包含模块分析增殖的数据。这些：包括FCSExpress（诺和软件，洛杉矶，CA）和FlowJo（树星公司，亚什兰，OR）。所有这些方案都使用一个非线性最小二乘分析，以迭代找到最适合的原始数据通过改变的位置，高度和SD（或宽度）的高斯峰，表示顺序的女儿代。增殖指数（PI）和前驱体频率（PF）是最常用的措施的增殖程度。 PI，定义由ModFit，是在测定过程中的细胞数量增加，类似于“刺激指数&#39;胸苷摄取试验的措施。 PF返回的馏分中的单元格的初始populat离子的刺激增殖。应注意，但是，当阅读文学术语有所不同套装软件之间（ 例如 ，，FlowJo和ModFit使用不同的定义和计算是什么意思“的扩散指数”）22。 用蛋白染料时也遇到了标签和膜染料的扩散分析与这里讨论的关键问题。例如，必须小心注意混合技术也可以观察到，随着排除死/死亡的细胞，为了获得均匀的分布和使用的CFSE的（ 图6）2-4,13,18,24区分女儿峰。选用合适的荧光染料的表型和生存能力评估也是很重要的，以避免过度的频谱重叠，无法识别抗体阳性细胞，特别是与可见光的发光蛋白染料如CFSE 2-4,11,13,16,18 </sup&gt;。减少跟踪染料的浓度降低赔偿的问题，在相邻光谱通道，但也限制了数量的细胞分裂，可监测的前子细胞与自体荧光强度开始重叠。另外，使用新的细胞示踪染料，如远红外发光CellVue干红葡萄酒（Sigma-Aldrich公司，圣路易斯，MO）或紫色的发光CellTrace紫（Life Technologies公司，大岛，NY）可能会降低赔偿的问题（ 图6）。最后，虽然膜的染料一般倾向于表现出较少的毒性11,26，它是可能的过与任一类的染料标记细胞。于是，它总是必要的验证，跟踪使用的染料的浓度并没有改变的细胞，以进行跟踪的功能（ 图6）3,13,16,18。

<table border="1"><tbody><tr><td> 试剂或设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 市售的 </td></tr><tr><td> 7 - 氨基放线菌素D </td><td> Sigma-Aldrich公司</td><td> A9400 </td><td></td></tr><tr><td>牛血清白蛋白（BSA） </td><td> Sigma-Aldrich公司</td><td> A4503 </td><td></td></tr><tr><td>牛胎儿血清（FBS）的</td><td>亚特兰大生物</td><td> S11150 </td><td></td></tr><tr><td> Hanks平衡盐溶液（HBSS） </td><td> Life Technologies公司</td><td> 14175-079 </td><td>钙和镁的自由，不加酚红</td></tr><tr><td>人IgG Cohn组分II和III球蛋白</td><td> Sigma-Aldrich公司</td><td> G-4386 </td><td></td></tr><tr><td>小鼠抗人CD3-FITC </td><td> BD Biosciences公司</td><td> 349201 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD4-APC </td><td> BD Biosciences公司</td><td> 340672 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD4 PECy7 </td><td> BD Biosciences公司</td><td> 348799 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD8-FITC </td><td> BD Biosciences公司</td><td> 347313 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD8-APC </td><td> CALTAG（Life Technologies公司） </td><td> MHCD0805 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD19-APC </td><td> CALTAG（Life Technologies公司） </td><td> MHCD1905 </td><tD&gt;饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD25-APC </td><td> BD Biosciences公司</td><td> 340938 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD127-PE </td><td> BD Biosciences公司</td><td> 557938 </td><td>饱和浓度由实验室滴定</td></tr><tr><td>小鼠抗人CD3 </td><td> eBiosciences </td><td> 16-0037-85 </td><td> 1.0毫克/毫升;磷酰基叠氮化物的免费</td></tr><tr><td>的小鼠抗人CD28 </td><td> eBiosciences </td><td> 16-0289-85 </td><td> 1.0毫克/毫升;磷酰基叠氮化物的免费</td></tr><tr><td> PBS </td><td> Gibco公司</td><td> 21300-058 </td><td></td></tr><tr><td> PKH26红色荧光的细胞连接器套件，包含10-3M PKH26在乙醇和稀释剂Ç </td><td> Sigma-Aldrich公司</td><td> PKH26GL-1KT 或 MINI26-1KT </td><td>第1步中的程序也适用箱PKH67或其他CellVue的染料</td></tr><tr><td>枣红远CellVue红色荧光细胞连接器套件，包含10-3M CellVue的干红葡萄酒在乙醇和稀释剂Ç </td><td> Sigma-Aldrich公司</td><td>的MINCLARET 1KT或MIDCLA​​RET的-1KT </td><td></td></tr><tr><td> 5 - （和6） - 羧基荧光素二乙酸酯，琥珀酰亚胺基酯（CFDA-SE） </td><td> Invitrogen公司（Life Technologies公司） </td><td> C34554 </td><td>非荧光;裂解膜酯酶形成荧光与氨基反应的羧基荧光素琥珀酰亚胺基酯（CFSE） </td></tr><tr><td> LIVE / DEAD绝不紫</td><td> Invitrogen公司（Life Technologies公司） </td><td> L34955 </td><td></td></tr><tr><td>无菌12×75毫米同轴 聚丙烯管帽</td><td> VWR </td><td> 60818-102 </td><td>提供了更好的膜染色效率（减少染料的吸附，减少细胞损失在上清愿望） </td></tr><tr><td> 12×75毫米的圆钢ð底部 聚苯乙烯管</td><td> Becton Dickinson公司</td><td> 21008-936 </td><td></td></tr><tr><td>流式细胞仪</td><td> BD生物科学</td><td> FACSCalibur LSRFortessa </td><td>任何能够检测FITC，PKH26，和7-AAD（例如488纳米;时间520纳米，567纳米和655纳米，分别）和APC前的流式细胞仪。 633-640 nm的时间。 660 nm）的</td></tr><tr><td>流式细胞仪</td><td> Beckman Coulter公司</td><td> LSRII青色</td><td>任何能够检测FITC，PKH26，和7-AAD（例如488纳米;时间520纳米，567纳米和655纳米，分别）和APC前的流式细胞仪。 633-640 nm的时间。 660 nm）的</td></tr><tr><td></td><td></td><td></td><td> 实验室制备 </td></tr><tr><td> 7 - 氨基放线菌素D， 集中持股</td><td> NA </td><td> NA </td><td>在PBS中的1毫克/毫升。冻结分装并储存于-20°C。 </td></tr><tr><td> 7 - 氨基放线菌素D， WOR王股票</td><td> NA </td><td> NA </td><td>在PBS的100微克/毫升，准备每天1毫克/毫升冷冻的股票。 </td></tr><tr><td>抗体块</td><td> NA </td><td> NA </td><td> HBSS + 10毫克/毫升人IgG Cohn组分II和III球蛋白+ 10毫克/毫升BSA。 </td></tr></tbody></table>

optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes

荧光细胞跟踪染料，与流量和图像流式细胞仪组合使用，是强大的工具，与不同类型的细胞在体外和体内研究的相互作用和命运1-5虽然使用这种染料的出版物有数千个，一些最常遇到的细胞追踪应用包括监测： <ol><li>干细胞和祖细胞静止，增殖和/或分化6-8 </li><li>抗原驱动的膜转移9和/或前体细胞的增殖3,4,10-18和</li><li>免疫调节和效应细胞的功能1,18-21。 </li></ol>市售的细胞示踪染料有很大的不同，它们的化学性质和荧光性质，但绝大多数落入两班的基础上他们的标记细胞的机制之一。 “膜染料”，以PKH26，高度亲脂性染料吨帽子分区稳定，但非共价结合到细胞膜1,2,11。 “蛋白质染料”，为代表的CFSE的氨基形成稳定的共价键的反应性染料与细胞蛋白4,16,18。每个等级都有其自身的优势和局限性。他们的成功使用的关键，特别是在多色多种染料的研究被用来跟踪不同类型的细胞，因此，实现最佳的使用每类2-4,16,18,24的了解的关键问题。 这里突出的协议包括三种常见的原因的穷人或不同的结果，当细胞示踪染料。这些是： <ol><li> 未能达到明亮，均匀的，可重复的标签 。这是一个必要的任何单元格追踪研究的出发点，但不同的变量时，用膜染料，而不是用蛋白染料或平衡结合，如抗体的试剂时，需要注意。 </li><li> 次优荧光合并ND /或不包括关键的补偿控制。跟踪染料的荧光一般是10 - 10 的3倍亮度比荧光抗体。因此，这是必需的验证，跟踪染料的存在下，不侵入的能力来检测正在使用的其他探针。 </li><li> 没有的峰值建模软件，以获得一个不错的选择 。这种软件可以定量比较不同人群或前体频率或其他指标的基础上刺激的增殖反应。获得一个不错的选择，但是，需要排除的死/死细胞，它可以扭曲染料稀释配置文件和匹配的基本特点，所观察到的染料稀释曲线模型的假设。 </li></ol>这里给出的实施例示出了如何将这些变量可以影响的结果，当使用膜的染料和/或蛋白质，以便监测细胞增殖。

Watch this Scientific Journal Video about Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes at JoVE.com

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

成功地利用监测免疫细胞的功能和增殖的细胞追踪染料涉及的几个关键步骤。我们描述的方法为：1）获得明亮，均匀，重现性好标签的膜染料; 2）选择荧光染料和数据采集条件; 3）选择一个模型来量化细胞增殖的基础上染料稀释。

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

optimized-staining-proliferation-modeling-methods-for-cell-division

Research

JoVE Journal

Biology

35.3K 次观看.  Roswell Park Cancer Institute. 成功地利用监测免疫细胞的功能和增殖的细胞追踪染料涉及的几个关键步骤。我们描述的方法为：1）获得明亮，均匀，重现性好标签的膜染料; 2）选择荧光染料和数据采集条件; 3）选择一个模型来量化细胞增殖的基础上染料稀释。

视频: Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

使用CyDye DIGE Fluor公司最小染料的细胞表面蛋白的选择性标签

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

科研

生物学

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

的三种不同方法对乳腺癌细胞株确定细胞增殖的比较

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

爪蟾卵母：显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

使用点击化学测量血管平滑肌细胞增殖

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...