作者想感谢凯特 Lavrich 在实验设计和稿件编辑方面的帮助。

1. 细胞的制备
注: 本程序描述永生细胞系的慢转导 (BEAS-2B;ATCC, 马纳萨斯, 弗吉尼亚州) 表达所需的记者 (roGFP2 或超级)。其他的细胞线/类型和/或基因转移的方法, 包括转染, 可以使用, 只要他们导致一个水平的记者表达足够的可视化足够数量的传感器表达细胞的每个领域的看法 (通常是 5-10 细胞)。如果使用转染方法, 则应在最终用于分析方法的盘中执行该过程 (例如, 将在同一盘中的细胞染到显微镜中)。下面介绍的步骤提供了准备在6孔细胞培养板中进行细胞 transductions 的细节。如果这个格式不是适当的大小为期望的应用, 其他船可以被替换。
<ol><li>在正常生长培养基中, 细胞生长到大约 40-60% 的汇合处。 注: 本研究采用人支气管上皮细胞系, BEAS-2B, 生长于角质生长介质 (魔芋)。</li>
	<li>就在转导之前, 用500µL 的无血清培养基中含有适量病毒的细胞取代生长培养基, 如公式所计算的: <img alt="Equation 1" src="/files/ftp_upload/56945/56945eq1.jpg"><ol>
<li>为 BEAS-2B 细胞的转导, 使用无血清角质形成基培养基 (KBM) 取代魔芋生长培养基在病毒孵化。 注: 上述计算是一个6井格式的一个细胞的单转导。如果需要, 执行 transductions 的几个井可以执行的调整公式乘以乘以数的转。对于慢 transductions, 使用在5和20之间的多重感染 (莫伊)。对于腺病毒 transductions, 使用100和500之间的莫伊。</li>
	</ol>
</li>
	<li>孵育细胞与病毒混合物在37° c 为 4 h, 再分配病毒微粒在盘中每30到60分钟以一个短暂摇摆或盘旋的运动。</li>
	<li>加入1毫升的完全生长培养基到盘中, 在37° c 下孵育 4-16 h。</li>
	<li>删除所有媒体并替换为全新的完全增长介质。</li>
	<li>继续生长和传代细胞, 根据需要进行扩张。 注意: 在 12-48 小时内, 细胞应该开始明显地表达传感器。如果使用慢向量进行转导, 则所需传感器的稳定表达式应在通道中继续。</li>
	<li>对于显微镜下的评估, 种子细胞到玻璃底的显微镜碟和成长到理想的汇合 (70-80%) 之前的成像。 注: 播种密度将取决于盘的大小、所用细胞的生长速率以及播种后的评估时间。例如, 种子 30万 BEAS-2B 细胞成一个35毫米的盘子, 在1天的生长后产生大约 70-80% 的汇合。</li>
</ol>2. 显微镜设置
注: 下面描述的协议是使用在404和 488 nm 上装有激光线的共聚焦显微镜进行的。对本协议中所描述的传感器进行荧光光谱评估的其他方法也应提供可行的数据。重要的是, 设备设置可以很大程度上取决于所使用的仪器的类型、年龄和条件;因此, 所提及的任何仪器价值可能并不具体于其他实验室使用的设备。
<ol><li>使用环境控制进行所有成像分析, 以保持适当的温度 (例如, 37 ° c)、湿度 (通常 > 95% 相对湿度) 和/或气体浓度 (例如, 5% CO2), 适用于整个实验的持续时间。</li>
	<li>如果使用较高的相对湿度值, 请保持任何可能接触湿化气氛的表面 (e. g, 显微镜物镜), 或略高于产生湿度的温度, 以防止冷凝。这可以通过使用一个客观加热器和/或加热胶带和一个适当的加热器控制来完成。</li>
	<li>打开所有显微镜组件, 并设置在488和 404 nm 的顺序励磁所需的所有设备, 其排放量在 510 nm。确保对光学配置的所有组件进行适当设置, 以便实时获取。</li>
	<li>在37° c、5% CO2大气压和 > 95% 湿度的条件下, 设置一个舞台顶部环境室以保持恒定温度。在开始图像采集之前, 在所有环境控制的初始设定后, 平衡环境室至少10分钟。 注意: 根据实验长度、细胞类型和所使用的暴露情况, 可以消除或调整环境条件。</li>
	<li>将细胞盘 (步骤 1.7) 放在环境室内的舞台顶端。</li>
	<li>用所需的物镜, 用目镜和白光找到细胞的焦平面, 确保正常的形态学。 注: 1.4 NA 60X 紫校正, 油浸没物镜通常使用, 这允许识别细胞内的舱室, 同时最大化的光学分辨率的共焦系统。</li>
	<li>在视场中检查细胞的荧光表达。在用合适的过滤器 (如荧光素异硫氰酸酯 (FITC)) 在广域荧光光照下对细胞进行可视化。在这一点上, 使用宽场照明, 而通过目镜看更方便, 因为它是更容易移动的菜选择一个领域的研究。一般情况下, 请选择包含至少5到10单元的视图字段, 它们表示传感器, 如绿色荧光所示。<ol>
<li>或者, 执行此评估 confocally 使用激光激励的波长是最兼容的传感器被表达 (488 nm 通常最有效)。 注: 由于其固有的荧光特性, 它将更难以可视化表达超高的细胞比那些表达 roGFP2 使用 FITC 或在 488 nm。然而, 仍然可以看到微弱的细胞。</li>
	</ol>
</li>
	<li>一旦找到了理想的视野, 关闭环境室。 注: 使用聚焦保持功能, 通常可在先进的显微镜支架上, 以方便维护一个稳定的焦平面在整个研究。</li>
	<li>设置购置参数, 以确保在所需的曝光期内对传感器进行最佳评估。以下是传感器响应共焦成像的建议和方法:<ol>
<li>调整激光功率为 488 nm 的励磁和发射在 510 nm。选择一个激光功率水平, 使细胞的可视化, 并保持这个常数之间的样品或复制菜肴。 注: 对于这项研究, 分别使用了488和 404 nm 激光线的12% 和1.5% 激光功率。</li>
		<li>使用采集软件的共焦控制, 以确保在调整 z 平面的同时, 在 488 nm 的情况下, 在单元中心 (z 轴) 上通过扫描, 对所选焦平面进行最大荧光发射强度的优化。在搜索导致最暴露的单元格的 z 平面时, 使用高增益设置会使这一点变得更容易。一旦找到适当的焦平面, 将增益返回到一个最理想的设置, 即在不 oversaturating 观察到的像素的情况下使用该报告的荧光。 注: 在实验过程中, 适合于发现和观察细胞的激光和增益设置完全取决于所使用的共焦系统。一般而言, 一旦在视场中发现了细胞, 建议使用少量的激光功率 (通常是≤20%), 因为使用高功率激光照射的细胞过度扫描可能会引起传感器检测到的氧化变化。</li>
		<li>利用增益来微调基线荧光。与 roGFP2, 建立基线附近的上限 (≈90% 相对强度) 的仪器没有过度饱和, 因为这些细胞将失去 510 nm 荧光诱导 488 nm 激发时, EGSH增加。相比之下, 488 nm 激发的荧光强度应低 (≈10% 相对强度) 在基线的超表达细胞, 因为这些细胞将获得 510 nm 荧光强度时, H2O2被检测到。</li>
		<li>重复步骤2.9.2 和2.9.3 与励磁在 404 nm 和发射在 510 nm。增益设置为 404 nm 励磁波长是相对于那些使用与 488 nm 励磁为每个传感器 (即低基线荧光 (≈10% 相对强度) 在404毫微米为 roGFP2, 高基线荧光 (≈90% 相对强度) 为 H2O2传感器)。 注: 一般而言, 荧光 (510 发射) 在 404 nm 激发将大大低于可获得的 488 nm 激发的细胞表达的 roGFP2 和超, 因为 404 nm 峰值是一个相对较小的励磁最大的两个传感器.</li>
	</ol>
</li>
</ol>3. 数据采集
<ol><li>设置采集软件, 按顺序激发两个激发波长 (先是 488 nm, 然后是 404 nm), 并在预期的时间间隔内, 在整个试验长度内收集 510 nm 的排放量 (例如, 每60捕获图像s 为60分钟)。<ol>
<li>或者, 如果在测试的异中已知 EGSH或 H2O2中更改的大致时间, 则在曝光前后手动获取图像。然而, 这可能会导致时间分辨率的丧失。</li>
	</ol>
</li>
	<li>对实验参数进行实时评估, 在现场选择至少 5-10 传感器表达单元, 并将其作为感兴趣的区域 ("roi"), 以监测其在实验过程中的荧光变化。 注意: 这一步是可选的, 并且可以在实验之后进行, 如果实时直接观察是不可取的, 或者是软件限制阻止了持续的监视。根据单元格的数量, 将单元格表示为 roi 的传感器的选择可能表示跨单元格的表达式级别范围。<ol>
<li>对于这些研究, 在 5 min 基线周期之后添加环境毒物 910-醌 (910-PQ) 或过氧化氢 (H2O2)。</li>
		<li>准备好所有的试剂以备以后在本协议中使用。 将二甲基亚砜中的 910 PQ 溶解到15毫米的浓度, 并在基底细胞介质中稀释, 产生250µM 的工作溶液。 此外, 准备一个水中过氧化氢的工作溶液, 将产生最终浓度为1mM 后注射。</li>
	</ol>
</li>
	<li>一旦确定了实验参数, 就开始时间课程的获取。在开始异曝光前建立至少5分钟 (或5数据点) 的基线周期。</li>
	<li>将细胞暴露在被调查的毒物中, 使用常规方法进行体外剂量。在水或其他合适的溶剂中制备可溶性化合物, 并直接注入介质。<ol>
<li>如果需要有机溶剂 (如二甲基亚砜或乙醇), 请将最终溶剂浓度保持在或低于0.1%。验证溶剂不产生对 EGSH或 H2O2生产的影响, 并在单独的一盘单元中使用车辆控制。</li>
		<li>对于溶解度较低的化合物, 在注射后使用微加入混合步骤。使用适当的载体气体混合物将气体暴露直接泵入环境室。</li>
	</ol>
</li>
	<li>在曝光期间监测荧光变化。根据需要进行后续注射。注意不要在注射过程中转移盘子, 这样在整个时间过程中, 同样的细胞被跟踪, 同时在同一焦平面上成像。</li>
	<li>在实验结束时, 将单元格暴露给适当的控件。对于这两种基因编码的荧光传感器, 添加特定浓度的化合物已知的氧化和减少这些传感器在一个尖锐的示范, 他们的比率反应。H2O2和糖 (德勤) 作为适当的控制来氧化和减少两个传感器。通常, 最终浓度为 100-1000 µM H2O2, 后跟 1-5 mM 糖 (德勤), 允许用户完全氧化和减少这些传感器。<ol>
<li>对于这些研究, 请使用 1 mm2O2 , 后跟 5 mm 的 "", 以确定传感器的最大响应。</li>
		<li>等待至少5分钟, 让传感器响应, 然后注入一个还原剂, 如, 以减少感光, 并返回到一个水平的荧光或附近的既定基线。 注意: 此步骤中的控件的使用对规范化至关重要, 在每个实验结束时都应执行, 以确定每个单元格中传感器的动态范围。对于 roGFP2, 在实验结束时也可以添加 aldrithiol。Aldrithiol 绕过 roGFP2 氧化还原继电器直接氧化传感器。这对于评估异曝光后的传感器功能很有用。</li>
	</ol>
</li>
</ol>4. 数据分析
<ol><li>如果尚未建立, 请在要分析的单元格周围绘制 roi。使用适当的软件来测量每个波长在整个时间过程中每个 ROI 的荧光强度。确保单元格不移动, 或在运行期间没有移出板。 注: roi 的建立是最容易完成的, 绘制一个封闭的区域, 遵循每个细胞的荧光表达模式。为了进一步帮助这个过程, 在确定细胞边界的努力中, 可以将细胞内的透射光 (或明) 图像叠加到荧光图像上。但是, 使用透射光图像需要用户在整个实验过程中捕获一个附加的通道 (图像集)。或者, 某些制造商将算法引入到他们的成像软件中, 使用特定的参数 (如强度阈值) 来建立更定量、更少主观的 roi 方法。</li>
	<li>将数据导出到所需的分析软件 (例如电子电子表格)。</li>
	<li>使用公式计算每个时间点的每个 ROI 的比率传感器响应: <img alt="Equation 2" src="/files/ftp_upload/56945/56945eq2.jpg"> <img alt="Equation 3" src="/files/ftp_upload/56945/56945eq3.jpg">
</li>
	<li>对于每个时间点, 平均所有 roi 的计算比率值。</li>
	<li>计算基线值 (将用于对数据进行规范化), 方法是将以前计算的比率值 (步骤 4.4) 的平均值 (在添加异 (例如, 前五个时间点) 之前收集。</li>
	<li>通过将每个值除以4.5 中计算的基线值, 将比率值正常化到步骤4.4。规范化比率将以基线值为1左右, 而在 EGSH或 H2O2以下注入的增加将导致比率增加。对诱导氧化改变的异的评估往往在基线值的 3-6 倍的范围内产生值。</li>
	<li>为了帮助比较实验中和跨试验的响应, 通过定义对控制氧化剂的响应 (例如, 1 mM2O2) 为 100%, 将规范化数据表示为最大传感器响应的百分比。 注意: 如果将数据表示为最大传感器响应的百分比, 则确保在实验中一致使用相同浓度的控制氧化剂是至关重要的。所有从活细胞成像分析得到的数据都可以根据研究者的判断选择常规的对智和群明智的统计分析。</li>
</ol>

本文所述的研究成果已由美国环境保护署国家卫生与环境影响研究实验室进行了审查, 并获准发表。本条的内容不应解释为代表机构政策, 也不提及商号或商业产品构成背书或推荐使用。

使用roGFP2 和超检测细胞内 EGSH和 H2O2的变化, 分别在以前的生理和毒理学研究中得到了很好的描述25,35,36 ,39,40,41,42,43。这里概述的协议报告了一种有效的方法来实现这些基?...

"氧化应激" 这一术语经常被称为毒理学中的一种机制, 但这一术语很少被具体描述。氧化应激可以指几种细胞内的过程, 包括活性氧的生成、自由基的损伤、抗氧化分子的氧化, 甚至是特定信号的激活。范围广泛的环境污染物1,2和药物代理3,4已被记录为通过异化合物本身的直接作用来诱导氧化应激5 ,6或次要地通过生产氧化剂种类作为蜂窝响应的一部分7、8、9、10。因此, 对毒理学有极大的兴趣, 能够准确地观察和描述导致不良结局的氧化过程。测量氧化应激的常规方法包括氧化生物分子的鉴定11,12,13,1415或抗氧化剂16 ,17,18,19,20, 或直接测量活性物种本身21,22,23, 24。但是, 这些方法通常需要蜂窝中断, 这通常会消耗样本, 消除空间分辨率, 并可能引入工件25。开发更灵敏和具体的方法来检测氧化剂种类和氧化应激标志, 广泛适用于研究异暴露的不利影响。
使用新一代基因编码的荧光传感器的活细胞显微镜已经成为监测细胞内氧化还原状态的有力工具。这些传感器通常是使用在病毒启动器的控制下通过转染或转导方法引入的载体来表达的。高表达效率是不必要的, 因为细胞表达的荧光传感器可以很容易地识别视觉。对于毒理学评估, 可以使用荧光显微镜观察表达这些传感器的细胞, 因为它们是实时接触异化合物的。这种实验设计允许在同一个单元中重复测量, 允许每个细胞的既定基线作为自己的控制。活细胞成像所提供的高时间分辨率非常适合于检测氧化事件, 特别是那些规模不大或瞬态性的。除了对它们的目标分子敏感和特异外, 这些传感器的荧光也可以用两种波长的光来激发。这种现象允许将荧光发射表达为一个比值, 它允许识别与真实传感器反应相关的信号变化, 如传感器表达的变化, 细胞厚度, 灯荧光探测器的强度、漂白和灵敏度26。使用荧光传感器的另一个优点是, 它们可以针对特定的蜂窝单元, 创建一个由常规方法25,26,27所无法比拟的空间分辨率级别。
一个以绿色荧光蛋白 (GFP) 为基础的基因编码的大家族被开发出来, 其特征是报告各种生理指标, 包括 pH 值、温度、钙浓度和 ATP/ADP 比值25 ,28,29,30,31。其中包括谷胱甘肽氧化还原电位 (EGSH) 和过氧化氢 (H2O2) 的传感器。虽然这些传感器是为应用于氧化还原生物学和生理学, 他们也已适应研究异诱导氧化应激。具体地说, 此处概述的协议描述了使用 EGSH传感器 roGFP2 和 H2O2传感器超级。
roGFP2 报告的氧化还原电位的细胞内还原和氧化谷胱甘肽 (GSH/GSSG) 通过一个氧化还原继电器涉及谷胱甘肽过氧化物酶 (GPx), 氧 (Grx), 谷胱甘肽还原酶 (GR) (图 1)25,32,33. 谷胱甘肽是主要的细胞抗氧化分子, 并存在于其还原形式 (GSH) 在 millimolar 浓度在胞25,34。虽然 EGSH尚未链接到任何功能结果, 但它被认为是细胞内氧化状态的重要指标34。GSSG 浓度的相对较小的增加会导致 EGSH的增加, 这是由 roGFP2 检测到的。同样重要的是, 在异暴露过程中使用 roGFP2 监测 EGSH , 可能会在氧化还原继电器和相关通路 (如戊磷酸盐分流器) 的几个点上揭示作用机理, 例如,图1)35。这里讨论的第二个传感器, 超, 是一个胞内 h2O2探针, 从插入黄色荧光蛋白 (YFP) 到细菌 H2O2敏感转录的规管领域中获得。因子 OxyR136。虽然它以前被认为是破坏性的活性氧中间体, H2O2越来越被认为是生理条件下的一个重要的细胞内信号分子37,38, 建议在毒理学中也存在用于 H2O2的无法识别的角色。例如, 由异曝光引起的过量的 H2O2可能是蜂窝信号中失调的前兆或生物的转移。
两个基因编码的荧光传感器都已被表达在几个既定的细胞线, 包括人表皮样癌细胞线 A431 和人支气管上皮细胞线 BEAS-2B, 以观察变化的 EGSH和 H2O2以响应各种毒理学暴露。这些包括气态污染物 (臭氧35)、微粒物质的可溶性组分 (12 醌39、40和锌41) 和镍纳米颗粒 (未发布的数据)。这些研究只代表了这两种传感器可能应用的一个小子集。理论上, 任何能够通过常规分子生物学技术接收和表达这些传感器的 DNA 的细胞类型都可以用来评估物质怀疑改变细胞氧化状态的影响。到目前为止, 这些传感器中的一个或多个已经表达了各种原和真核生物, 包括几个哺乳动物, 植物, 细菌和酵母细胞类型25,26,36,42。EGSH和 H2O2传感器的读数是在激发时在 510 nm 发出的荧光强度随488和 404 nm 光的变化。该方法广泛适用于荧光平台, 包括各种类型的显微镜 (共焦和广域) 和平板阅读器。这里提出的方法允许对细胞内 EGSH和 H2O2在体外毒理学系统的敏感和具体观察。

<table><tbody><tr><td>roGFP2 Plasmid</td><td>University of Oregon</td><td>N/A</td><td>Generous gift of S.J. Remington</td></tr><tr><td>HyPer Plasmid</td><td>Evrogen</td><td>FP941</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma Aldrich</td><td>H1009</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich</td><td>10708984001</td><td></td></tr><tr><td>9,10-Phenanthrenequinone</td><td>Sigma Aldrich</td><td>156507</td><td></td></tr><tr><td>Black Wall Glass Bottomed Dishes</td><td>Ted Pella</td><td>14029-20</td><td></td></tr><tr><td>BEAS-2B cell line</td><td>American Type Culture Collection</td><td>CRL-9609</td><td></td></tr><tr><td>Keratinocyte Basal Medium</td><td>Lonza</td><td>192151</td><td></td></tr><tr><td>Keratinocyte Growth Medium BulletKit</td><td>Lonza</td><td>192060</td><td></td></tr><tr><td>Excel&amp;nbsp;</td><td>Microsoft Office Suite</td><td>N/A</td><td></td></tr><tr><td>NIS-Elements AR Imaging Software</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Nikon C1si Confocal Imaging System</td><td>Nikon</td><td>N/A</td><td></td></tr></tbody></table>

imaging approaches to assessments of toxicological oxidative stress using genetically-encoded fluorogenic sensors

虽然氧化应激是一种常用的毒理学机制, 但传统的研究方法存在许多缺陷, 包括样品的破坏、潜在的伪影的引入, 以及缺乏反应的特异性涉及的物种。因此, 目前需要在毒理学领域的非破坏性的, 敏感的, 和具体的方法, 可以用来观察和量化细胞内氧化还原摄动, 更常见的称为氧化应激。在这里, 我们提出了一种方法, 使用两个基因编码的荧光传感器, roGFP2 和超, 将用于活细胞成像研究, 以观察异诱导的氧化反应。roGFP2 均衡与谷胱甘肽氧化还原电位 (EGSH), 而超高直接检测过氧化氢 (H2O2)。这两种传感器都可以通过转染或转导的方式表达成各种细胞类型, 并且可以针对特定的细胞室。最重要的是, 使用这些传感器的活细胞显微镜提供了高的空间和时间分辨率, 这是不可能使用传统的方法。在 510 nm 监测的荧光强度的变化作为两个基因编码的荧光传感器的读数, 当连续兴奋 404 nm 和 488 nm 光。该属性使两个传感器比率, 消除了普通的显微工件, 并纠正了传感器在细胞之间的表达差异。这种方法可以应用于各种荧光平台, 能够激发和收集规定波长的排放, 使其适合于共焦成像系统、常规的广域显微镜和平板读者.两种基因编码的荧光传感器都被用于各种细胞类型和毒理学研究, 以实时监测细胞 EGSH和 H2O2生成。这里概述了一个标准化的方法, 广泛适用于细胞类型和荧光平台的应用 roGFP2 和超高活性细胞毒理学评估的氧化应激。

在检测 EGSH和胞内 H2O2中使用 roGFP2 和超高的方法时, 已经很好地描述了以前的25、36、42、43 , 并在此演示。在基线上表达 roGFP2 的细胞的共焦图像, 并在 H2O2和数字显示之后添加了图 2。然后按照《议定书》4节的描?...

Watch this Scientific Journal Video about Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors at JoVE.com

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

这篇手稿描述了使用基因编码的荧光记者在一个应用的活体细胞成像检查异诱导氧化应激。这种实验方法提供了无与伦比的时空分辨率、灵敏度和特异性, 同时避免了用于检测毒理学氧化应激的常规方法的许多缺点。

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 ...

imaging-approaches-to-assessments-toxicological-oxidative-stress

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

7.4K 次观看.  University of North Carolina at Chapel Hill. 这篇手稿描述了使用基因编码的荧光记者在一个应用的活体细胞成像检查异诱导氧化应激。这种实验方法提供了无与伦比的时空分辨率、灵敏度和特异性, 同时避免了用于检测毒理学氧化应激的常规方法的许多缺点。

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

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

筛选法测定氧化应激在猫的星形胶质细胞的细胞线，G355 - 5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

科研

神经科学

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. ...

生物化学

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 ...

生物工程

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

生物学

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (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, ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

用基因编码荧光传感器评估毒理学氧化应激的成像方法

摘要

探索更多视频

筛选法测定氧化应激在猫的星形胶质细胞的细胞线，G355 - 5

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

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

在斑马鱼胚胎氧化应激分析

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

使用高含量显微镜的细胞氧化还原分析

C. Elegans生理应激反应的测量

用基因编码荧光传感器评估毒理学氧化应激的成像方法

摘要

探索更多视频

筛选法测定氧化应激在猫的星形胶质细胞的细胞线，G355 - 5

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