This research was supported by the University of Antwerp (TTBOF/29267, TTBOF/30112), the Special Research Fund of Ghent University (project BOF/11267/09), NB-Photonics (Project code 01-MR0110) and the CSBR (Centers for Systems Biology Research) initiative from the Netherlands Organization for Scientific Research (NWO; No: CSBR09/013V). Parts of this manuscript have been adapted from another publication1, with permission of Springer. The authors thank Geert Meesen for his help with the widefield microscope.

对于NHDF细胞和使用材料文件中指定的多孔板，描述了下面的方案。有关工作流程的总体概述，请参见图1 。 1.试剂的制备<ol><li>通过用10％v / v胎牛血清（FBS）和100IU / mL青霉素和100IU / mL链霉素（PS）补充Dulbecco&#39;s Modified Eagle Medium（DMEM）来制备完整培养基。对于500 mL完全培养基，加入50mL FBS和5 mL PS至445 mL DMEM。 </li><li>通过用20mM HEPES补充汉克平衡盐溶液（含镁和钙，但不含酚红）制备HBSS-HEPES（HH）成像缓冲液。对于500 mL HH，加入10 mL 1M HEPES储备溶液至490 mL HBSS。验证pH值，如果需要，调整pH值为7.2。 </li><li> 通过将50μgCM-H 2 DCFDA冻干粉末溶解在86.5℃下制备1mM CM-H 2 DCFDA储备溶液，＃181; L无水DMSO。通过涡旋或上下移液混合，并在棕色微量离心管中制备20μL等分试样。储存于-20°C的黑暗中，并在1周内使用。如果储存在N 2大气保质期内可以增加至少1个月。 <ol><li>通过将25mg TMRM粉末溶于50mL无水DMSO中制备1mM TMRM储备溶液。通过涡旋或上下移液混合，并在棕色微量离心管中制备10μL等分试样。储存于-20°C的黑暗中。该解决方案至少稳定一年。 </li></ol></li><li>通过直接从70％储备溶液（10μL/ 96孔板）中移取体积，为每个实验准备新鲜的叔丁基过氧化物（TBHP）储备（〜7M）的等分试样。将此等分试样保持在4°C直到进一步使用。 </li><li>通过在1x HH-bu中稀释两次二抗（Alexa Fluor 488驴抗兔和CY3驴抗兔）来制备抗体工作溶液（AB）1000次FFER。 </li></ol> 2.设置显微镜和采集协议（±15分钟） 注意:使用配有自动化舞台和百叶窗的宽视野显微镜以及使用20X空气计划校正物镜（NA = 0.75）和EM-CCD摄像机的基于硬件的自动对焦系统执行图像采集。当首次设置测定时，使用含有根据方案说明书染色的对照细胞的测试板来校准XY阶段并优化采集设置。如果采集设置已经确定，可以使用空板进行校准。 <ol><li>将目标降低到绝对基准水平，并校准XY阶段以下软件指令。 </li><li>确保安装了正确的过滤器立方体。为了可视化CM-H 2 DCFDA，请使用带有472/30 nm激发带通滤光片的标准GFP滤光片，495 nm截止二向色ic镜和520/35 nm带通发射滤光片。对于TMRM，使用带有540/25 nm带通激励滤光片，565 nm截止分色镜和605/55 nm带通发射滤光片的TRITC滤光镜立方体。 </li><li> 使用采集软件创建成像协议。 <ol><li>从软件中提供的可用板列表中选择正确类型的多孔板（制造商和代码）。或者使用板和孔尺寸定义您自己的多孔板格式。 </li><li>按照软件说明对准井板， 例如定义四个外角井的两个角。此步骤适用于摄像机方向的变化。 </li><li>选择需要采集的井。如果软件中没有此选项，请使用与所选孔对应的一组手动定义的XY位置。 </li><li>优化采集设置（曝光时间，灯强度，EM增益）e两个通道分别使用测试板。荧光激发光本身引起ROS时，可最大限度地减少曝光和强度。但是，在TBHP治疗前，确定基底CM-H 2 DCFDA和TMRM信号与背景比至少为2，TBHP治疗后没有饱和。采集设置极大地取决于所使用的显微镜​​设置和细胞类型，但作为参考，当使用130W的金属卤化物灯泡作为光源和根据方案说明书染色的NHDF细胞时的指示性设置如下:对于CM- H 2 DCFDA和TMRM使用200 ms的曝光时间和ND滤镜8，分别结合15（13 MHz; 14位）和4（27 MHz; 14位）的EM增益。一旦针对特定的设置和单元格类型进行了优化，可以跳过此步骤。 注意:在整个成像过程中采集设置必须保持一致。对于大规模，多天的实验，灯具必须通过定期的质量控制来保证健康。 </li><li>定义采集协议，由连续的λ（波长）采集组成。选择首先要采集的CM-H 2 DCFDA通道，以最小化测量前的光照。 </li><li>定义一个井盘循环，使用2.3.4中定义的采集协议获取位于井选择的每个井的中心周围的4个有规律间隔的非重叠图像。选择蜿蜒图像采集， 即从左到右，从B02到B11，然后从右到左从井C11到C02等等（ 图2A ）。与左至右图像采集相比，这节省了时间。如果软件中没有此选项，请调整在2.3.3中创建的XY位置的自定义集，以进行此成像模式。 </li><li>保存成像位置的XY坐标（ 例如 ，在单独的xml文件中），以便轻松重新设计在显微镜重新校准的情况下。如果该测定的读数必须与相同细胞的事后免疫荧光（IF）染色相关，这一点尤为重要。 </li></ol></li></ol> 3 。 96孔板中的接种细胞（45-90分钟，取决于不同细胞系的数目） <ol><li>在无菌环境中工作，如2级生物安全柜和戴手套。 </li><li>使用70％v / v乙醇在蒸馏水中净化所有表面和材料。 </li><li>从培养箱中取出90％汇合细胞培养物的细胞培养瓶，并将其置于生物安全柜中。 </li><li>用PBS 1x洗涤细胞两次。 </li><li>在细胞上加入适量的0.05％胰蛋白酶-EDTA溶液，确保覆盖完整的细胞表面（ 例如 T25烧瓶1 mL），37℃和5％CO 2孵育2 min。 </li><li>如果所有细胞都分离（用显微镜检查）<html> ），加入培养基（DMEM + 10％FBS + 1％青霉素 - 链霉素; T25烧瓶中±4mL）灭活胰蛋白酶-EDTA溶液。 </li><li>在室温下以300xg离心5分钟。 </li><li>弃去上清液，将细胞沉淀重悬于培养基中。确定每种细胞类型的培养基的量以获得与细胞计数相容的细胞浓度。通常，90％的汇合的T25烧瓶的NHDF含有约1-1.5百万个细胞，将其重新悬浮在3-4mL的培养基中。 </li><li>使用细胞计数室或库尔特计数器计数细胞。 </li><li>在具有薄的连续聚苯乙烯或玻璃底部（黑色以避免成像期间相邻孔之间的散射和串扰）的黑色96孔板的每个内部60孔中种子8,000-10,000个细胞。当使用不同的条件/处理/细胞系时，将它们的播种位置均匀地分布在板上以便最小化板效应（图2A），除了B01和A01井外，外部井不使用，因为它们更容易发生边缘效应。 </li><li> B01种子8,000-10,000个细胞。这个井将用于焦点调整，就在图像采集之前。 </li><li>用空气填充空的外部井，以使井和环境之间的梯度（温度，湿度等 ）最小化。 </li><li>轻轻敲打板三次，然后放回孵化器，以避免细胞生长在斑块中。 </li><li>培养细胞24小时，或达到汇合程度约。 70％。 </li><li>将实验的治疗信息保存到名为"Setup.xlsx"的电子表格中。该文件应包含四列，并将用于在数据分析期间将处理与井和图像信息进行链接。四列是:"好"，"待遇数"，"待遇"和"控制"（每排一行）。每个治疗都加以联系具有独特的治疗次数，在数据可视化期间用于确定地块X轴处理的顺序。 control-column指定用作当前行处理控件的处理。典型实验布局和相应设置文件的插图如图2所示 。 </li></ol> 4.用CM-H 2 DCFDA和TMRM装载细胞（±45分钟） 注意:实验当天可以在无菌环境（生物安全柜）中进行细胞处理，但这并不是强制性的，因为细胞将在测定后直接丢弃或固定。 <ol><li>将HH缓冲液加热至37°C。 </li><li>通过在HH缓冲液中稀释1mM储备溶液50次（加入490μLHH缓冲液至10μLTMRM储备溶液的1份），制备20μMTMRM工作溶液。 </li><li> 准备一个负载具有2μMCM-H 2 DCFDA和100​​nM TMRM的溶液。为此，在HH缓冲液中稀释1mM CM-H 2 DCFDA储备溶液500x和20μMTMRM工作溶液200x。 <ol><li>通常，对于60孔，通过加入15μL的CM-H 2 DCFDA和37.5μL的TMRM溶液至7447.5μL的HH中，制备7.5mL的加载溶液。 </li></ol></li><li>通过在单个流体运动中将板翻转而将细胞从细胞中丢弃。 </li><li>使用多通道移液管（100μL/孔），用HH缓冲液轻轻洗涤细胞两次。在洗涤步骤之间，通过在单个流体运动中倒转板来舍弃HH缓冲液。 </li><li>通过向每个孔中加入100μL的加载溶液，再次使用多通道移液管，将细胞装入CM-H 2 DCFDA和TMRM。在黑暗中，室温孵育25分钟。不要忘了B01。 </li><li>在这25分钟内，准备氧化剂TBHP的工作溶液，并确保显微镜和附件硬件已打开。 <ol><li>准备工作溶液I:将7M储备溶液稀释至70mM至100mM（在690μLHH缓冲液中10μL储备液）。 </li><li>准备工作溶液II:稀释WS I 100x至1mM（10μLWS I，在990μLHH缓冲液中）。 </li><li>准备工作解决方案III:稀释WS III 25x至40μM（对于60孔，在7200μLHH缓冲液中加入300μLWS II）。 </li></ol></li><li> 25分钟后，如前所述，用100μLHH缓冲液再洗两次细胞。 </li><li>向所有60个内孔中加入100μLHH缓冲液。 </li></ol> 5.首次实时成像测量基础ROS水平和线粒体形态功能（±15分钟） <ol><li>确保采集软件正常运行，并加载成像协议。 </li><li>在显微镜上安装板，打开硬件sed自动对焦系统，并使用B01来使用TMRM通道调整自动对焦偏移。由于该过程引起CM-H 2 DCFDA信号强度的增加，所以从下游图像分析中排除了该井。 </li><li>运行成像协议。 </li></ol> 6.添加TBHP后测量诱导的ROS水平（±20分钟）后的第二次实时成像<ol><li>小心地从显微镜上取出96孔板。 </li><li>使用多通道移液管向每个孔中加入100μLTBHP WS III（40μM）（这导致孔中的20μMTBHP浓度）。该化合物用作CM-H 2 DCFDA染色的内部阳性对照（信号应该升高）以及测量诱导的ROS水平的手段。 注意:也可以使用H 2 O 2代替TBHP，但该化合物的稳定性较差，因此不太可靠。 </li><li>等待至少3分钟，以使TBHP完全反应CM-H 2 DCFDA。 </li><li>在此期间，向井A01加入100μL抗体工作溶液（1 / 1,000）。 </li><li>将显微镜放回显微镜上并再次使用B01进行对焦。 </li><li>使用相同的成像协议获取与第一次成像轮次相同的位置。 </li><li>使用标准化命名法将单个文件夹中的获取数据集导出为单个文件夹，其中包括引用板，TBHP之前或之后处理，以及以下划线分隔的字段和通道。板1，预TBHP处理，井B02，场1和通道1的"P01_Pre_B02_0001_C1"。在图像分析期间将使用该信息（ 例如 ，选择适当的分割设置）以及数据分析期间（用于连接分析数据用正确的治疗方法）。 </li><li>使用采集协议，在井A01中心周围的所有四个位置获取两个通道的平坦场图像。节省em作为与其他图像相同文件夹中的单独tiff文件使用以下标准化命名:板1，场1和通道1的"P01_FF_A01_0001_C1"。确保信号处于动态范围内;在饱和的情况下，使用较低浓度的抗体工作溶液。 </li><li>丢弃板或保存进一步处理。 注意:不用从显微镜上移除板，并使用多通道移液管添加TBHP溶液，而是可以在显微镜平台上安装自动移液器，并与采集软件连接，以便在接收到触发时起作用。这样可以在首次采集后直接向每个井添加TBHP，然后再移动到下一个井。这样，当第一次成像轮次完成时，第二个成像轮可以立即开始，所有的井将与TBHP具有相同的孵化时间。 </li></ol>图像处理与分析（±30分钟/96孔板） 注意:所有图像处理都在FIJI（http://fiji.sc）中执行，该版本是ImageJ免费软件的封装版本。编写了专门的脚本，用于自动分析细胞内ROS和线粒体信号，以及形态参数（RedoxMetrics.ijm，可根据要求提供）。基础算法在Sieprath 等人 1 。 <ol><li>确保FIJI已安装并运行。 </li><li>启动FIJI并安装宏集（插件 - &gt;宏 - &gt;安装...）。这将调用一些新的宏命令以及一组操作工具来优化分析设置， 如图3A所示 。 </li><li> 打开设置界面，通过单击"S"按钮设置分析设置（ 图3B ）。 <ol><li>选择图像类型，通道数和使用的孔用于获取平面图像。 </li><li>指示哪个通道包含细胞（CM-H 2 DCFDA通道）或线粒体（TMRM通道），并根据图像质量调整每个通道的预处理和分割参数（ 图3B ）。勾选"背景"和"对比度"复选框，分别执行背景减除或对比度限制的自适应直方图均衡16 。定义高斯模糊细胞的西格玛和拉普拉斯线粒体增强。选择自动阈值算法并填写大小排除限制（以像素为单位）。如果选择了固定的阈值而不是自动阈值算法，请填写上限阈值。 </li><li>分别通过打开它们并单击菜单中的"C"或"M"按钮分别测试获取的数据集的几个选定图像上的分割设置，分别进行细胞或线粒体细分，并根据需要调整设置。示例结果如图3C所示 。 </li></ol></li><li>通过点击"＃"按钮并选择带有图像的文件夹，对所关心的文件夹运行批处理分析。每个文件夹，这将产生一个新的"输出"目录，每个图像包含各个ROI集（zip文件）和结果文件（.txt）。对于两个通道，结果文件包含强度和形态描述符。 CM-H 2 DCFDA通道（单元格）结果文件包含每个描述符一个图像内的组合ROI的平均值。对于TMRM通道，结果文件每个描述符包含每个单独分割的线粒体ROI的值。 </li><li>批量分析后，通过点击"V"按钮，目视验证"验证堆栈"上的分割效果，所有图像的超级堆叠具有各自的ROI覆盖。这样，/低分辨率，图像中的焦点失真图像或灰尘/光纤可以快速发现。在数据质量控制期间可以进行进一步的策划（cfr。§8）。 </li></ol> 8.数据分析，质量控制（QC）和可视化原始数据的处理和分析使用R统计免费软件（http://www.rproject.org - 版本3.3.2）和RStudio（http://www.rstudio.com/ - 版本1.0.44）完成。为了快速获取和可视化结果，已经设想出了一个直观的Shiny应用程序17 （可根据要求提供），它集成和可视化加热图和箱体图中的数据，并进行统计分析。一般而言，工作流程包括两个连续的步骤。首先，每96孔板处理和检查数据以检测异常数据点。其次，将给定实验的所有板的策划数据组合并使用非参数多分析变量测试18和主成分分析。 <ol><li>确保R和RStudio已安装并可操作。 </li><li>启动RStudio。 </li><li>打开RedoxMetrics闪亮的应用程序并运行应用程序（选择在外部浏览器中运行）。 </li><li>在"输入"页面上，选择RedoxMetrics.ijm中的结果文件所在的目录。 </li><li>确保"Setup.xlsx"（在步骤3.15中创建）也存在于此目录中。 </li><li> 结果文件和设置信息自动导入，重新排列和可视化。 <ol><li> "实验设置"页面显示实验的布局。使用此页面进行验证。 </li><li>下一页"每板结果"分别以彩色编码的多孔板布局以及带有标有名称和图像编号的异常值的框图显示每个板的数据。后者允许轻松检查和识别异常数据点的异常（与特定治疗测量的平均值相比极高或较低的值）， 见图4 ）。 使用此信息，验证与异常点相对应的图像。如果检测到异常，则必须从分析中移除。当图像的（部分）失焦或高度荧光粉尘粒子干扰适当的分割时，大多数异常是由不正确的分割引起的。可以使用验证堆栈工具快速发现极端情况（步骤7.5），但在此步骤中会发现更多的微妙事件。当没有明显的或技术上的原因找到异常值时，图像不应该从分析中去除。 </li><li>可选:创建一个名为"Drop.xlsx"的新电子表格，只有一列名为"Drop"。在此列中列出所有图像的文件名（包括".tif"扩展名）必须从分析中删除（在步骤7.5或步骤8.6.2中确定）。使用"输入"页面上传此文件。 </li><li> "结果整体实验"页面显示了所有板块的结果。如果下载文件被上传，则该文件用于从分析中删除指定的数据点。 对于每个参数，每个参数根据各自的控制进行标准化。然后将来自所有板的数据组合，随后来自nparcomp包18的非参数多变量测试。如果仅比较两种处理，则进行非参数的Behrens-Fisher问题的两个样本测试。对于超过2种治疗，使用非参数对比度多重比较检验。结果使用boxplots可视化。 </li><li> "集群分析"页面显示了主成分分析（PCA - R core&#39;stats&#39;包）的结果。 5个参数的数据（basal和诱导的ROS和线粒体膜电位，大小和圆形度）组合以基于敏感的氧化还原曲线区分不同的治疗。为此，执行主成分分析（PCA）（R core&#39;stats&#39;package）。结果使用biplot（ggbiplot软件包 - http://github.com/vqv/ggbiplot）可视化。 </li><li>使用"下载数据"页面下载包含已处理和重新排列的数据的后端数据帧，以便可以将其重新用于更高级的数据可视化或统计分析。 注意:典型的陷阱和潜在的解决方案列在表1中 。 </li></ol></li></ol>

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</ol>

作者指出，没有竞争的经济利益或其他利益冲突。相应的作者还确保所有作者被要求披露任何和所有的利益冲突。

本文介绍了用于同时定量NHDF中细胞内ROS水平和线粒体形态功能的高含量显微镜方法。通过对SQV处理的NHDF的案例研究证明其性能。结果支持早期的文献证据，其中在用1型HIV蛋白酶抑制剂治疗后已经观察到ROS水平升高或线粒体功能障碍，尽管在单独的实验19,20,21,22,23,24,25中。重要的区别是所描述的测定能够在相同的活细胞中同时测量这些参数，以及形态学数据。这种方法的主要优点在于它们在空间?...

<html>通过ROS产生和ROS消除系统之间的动态相互作用，精细调节细胞内ROS的浓度。两者之间的不平衡引起氧化应激状态。线粒体的主要来源之一是线粒体1 。鉴于它们在细胞呼吸中的作用，它们负责大量的细胞内超氧化物（O 2 · - ）分子2 。这主要是由于电子传输链的复合物1在强的负内线粒体膜电位（Δψm）， 即线粒体超极化的条件下，电子泄漏到O 2 。另一方面，线粒体去极化也与增加的ROS生产相关，指向多种作用模式3,4,5 ，&gt; 6，7，8 。此外，通过裂解融合机理的蛋白质的氧化还原修饰，ROS共调节线粒体形态9。例如，碎片与增加的ROS产生和凋亡相关， 10,11 ，而丝状线粒体已经与营养物质饥饿和保护相关mitophagy 12 。鉴于细胞ROS和线粒体形态功能之间的复杂关系，两者应理想地在活细胞中同时量化。为了做到这一点，基于自动化宽视野显微镜和用荧光探针CM-H 2 DCFDA（ROS）和TMRM（线粒体Δψm和形态）染色的贴壁细胞培养物的图像分析开发了高含量成像测定。高含量成像是指提取sp关于使用多个互补标记和自动图像分析的细胞表型的信息丰富（ 即大量描述性特征）。当与自动显微镜组合时，可以并行筛选许多样品（ 即高通量），从而增加测定的统计学功能。实际上，协议的主要资产是它允许在同一个信元中同时量化多个参数，而这对于大量的信元和条件来说是可以的。 协议分为8个部分（以下协议详细描述）:1）96孔板中的播种细胞; 2）储备溶液，工作溶液和成像缓冲液的制备; 3）设置显微镜; 4）用CM-H 2 DCFDA和TMRM装载细胞; 5）首次实时成像测量基础ROS水平和线粒体形态功能; 6）加入叔丁基后进行第二次活体成像过氧化物（TBHP）测量诱导的ROS水平; 7）自动图像分析; 8）数据分析，质量控制和可视化。 该测定法最初是针对正常人皮肤成纤维细胞（NHDF）开发的。由于这些细胞是大而平坦的，它们非常适合于评估2D宽幅图像13,14 中的线粒体形态。然而，通过轻微修改，该方法适用于其他贴壁细胞类型。此外，在CM-H 2 DCFDA和TMRM的组合之后，工作流程符合各种具有不同分子特异性的荧光染料对1,15。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM)</td><td>ThermoFisher Scientific</td><td>T668</td><td></td></tr><tr><td>CM-H2DCFDA (General Oxidative Stress Indicator)</td><td>ThermoFisher Scientific</td><td>C6827</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma)Aldrich</td><td>D8418</td><td></td></tr><tr><td>MatriPlate 96-Well Glass Bottom MicroWell Plate 630 &amp;micro;L-Black 0.17 mm Low Glass Lidded</td><td>Brooks life science systems</td><td>MGB096-1-2-LG-L</td><td></td></tr><tr><td>HBSS w/o Phenol Red 500 mL</td><td>Lonza</td><td>BE10-527F</td><td></td></tr><tr><td>DMEM high glucose with L-glutamine</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>Phosphate Bufered Saline (PBS) w/o Ca and Mg</td><td>Lonza</td><td>BE17-516F</td><td></td></tr><tr><td>HEPES 1 M 500 mL</td><td>Lonza</td><td>17-737F</td><td></td></tr><tr><td>Trypsin-Versene (EDTA) Solution</td><td>Lonza</td><td>BE17-161E</td><td></td></tr><tr><td>Cy3 AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson</td><td>711-166-152</td><td>Antibody used for acquiring flat-field image</td></tr><tr><td>Alexa Fluor 488 AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson</td><td>711-546-152</td><td>Antibody used for acquiring flat-field image</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nikon Ti eclipse widefield microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Perfect Focus System (PFS)</td><td>Nikon</td><td></td><td>hardware-based autofocus system</td></tr><tr><td>CFI Plan Apo Lambda 20X objective</td><td>Nikon</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NIS Elelements Advanced Research 4.5 with JOBS module</td><td>Nikon</td><td></td><td>This software is used to steer the microscope and program/perform the automatic image acquisition prototocol</td></tr><tr><td>ImageJ (FIJI) Version 2.0.0-rc-43/1.50g</td><td></td><td></td><td></td></tr><tr><td>RStudio Version 1.0.44</td><td>Rstudio</td><td></td><td></td></tr><tr><td>R version 3.3.2</td><td></td><td></td><td></td></tr></tbody></table>

cellular redox profiling using high-content microscopy

活性氧（ROS）调节基本细胞过程，包括基因表达，迁移，分化和增殖。然而，过量的ROS水平诱导氧化应激状态，伴随着对DNA，脂质和蛋白质的不可逆的氧化损伤。因此，ROS的定量为细胞健康状况提供了直接依据。由于线粒体是ROS的主要细胞来源和靶标，所以在同一细胞中线粒体功能和ROS产生的联合分析对于更好地了解病理生理条件的互连至关重要。因此，开发了基于高含量显微镜的策略，用于同时定量细胞内ROS水平，线粒体膜电位（ΔΨm）和线粒体形态。它是基于自动化的宽场荧光显微镜和生物贴壁细胞的图像分析，生长在多孔板和staine中d与细胞可渗透的荧光报告分子CM-H 2 DCFDA（ROS）和TMRM（ΔΨm和线粒体形态）。与荧光测定法或流式细胞术相反，该策略允许在实验刺激前后具有高时空分辨率的个体细胞水平的亚细胞参数的定量。重要的是，该方法的基于图像的性质允许除了信号强度之外还提取形态学参数。组合特征集用于探索和统计多变量数据分析，以检测亚群，细胞类型和/或治疗之间的差异。在此，提供了测定的详细描述，以及证明其在化学扰动之后在细胞状态之间明确区分的潜力的示例性实验。

该测定已经使用几个对照实验进行了基准测试，其结果在Sieprath et al。 1 。简而言之，CM-H 2 DCFDA和TMRM的荧光响应分别定量分析了细胞内ROS和Δψm的变化，以确定其动态范围。对于CM-H 2 DCFDA，当用10μM至160μM范围内的增加的TBHP浓度处理时，NHDF显示出荧光信号的线性增加。同样，对于TMRM，当用1〜10μg/μL范围内增加浓度的寡霉素（其诱?...

Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

Cellular Redox Profiling Using High-content Microscopy

本文提出了一种高含量显微镜工作流程，用于同时定量细胞内ROS水平，以及线粒体膜电位和形态 - 共同称为线粒体形态功能 - 使用细胞透过性荧光报告分子5-（和 - 6） - 氯甲基-2&#39;，7&#39;-二氯二氢荧光素二乙酸酯，乙酰酯（CM-H 2 DCFDA）和四甲基罗丹明甲酯（TMRM）。

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

cellular-redox-profiling-using-high-content-microscopy

Research

JoVE Journal

Biology

10.9K 次观看.  University of Antwerp. 本文提出了一种高含量显微镜工作流程，用于同时定量细胞内ROS水平，以及线粒体膜电位和形态 - 共同称为线粒体形态功能 - 使用细胞透过性荧光报告分子5-（和 - 6） - 氯甲基-2'，7'-二氯二氢荧光素二乙酸酯，乙酰酯（CM-H 2 DCFDA）和四甲基罗丹明甲酯（TMRM）。 

视频: Cellular Redox Profiling Using High-content Microscopy

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

生物化学

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

生物学

Autofluorescence Imaging to Evaluate Cellular Metabolism

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.

This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.

自发荧光成像评估细胞代谢

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced ...

生物工程

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

使用双氟比度显微镜测量巨无素体的pH值、重度化学和降解能力

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

免疫与感染

在肝癌细胞中使用二氢乙锭进行新型 ROS 定量

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

作者聚焦：肝细胞系中细胞内 ROS 水平的高通量测量

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

使用高含量显微镜的细胞氧化还原分析

摘要

探索更多视频

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

利用同位素标记的质谱剖析巯基氧化还原蛋白质

自发荧光成像评估细胞代谢

使用双氟比度显微镜测量巨无素体的pH值、重度化学和降解能力

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

使用高含量显微镜的细胞氧化还原分析

摘要

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用基因编码荧光传感器评估毒理学氧化应激的成像方法

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