dihydroethidium &#40;dhe&#41; staining of hepatocarcinoma cells for the quantification of reactive oxygen species &#40;ros&#41;

在肝癌细胞中使用二氢乙锭进行新型 ROS 定量

Reactive oxygen species (ROS) are a group of naturally occurring, highly reactive, and temporally unstable chemical radicals formed as a part of the normal cellular metabolism in&#160;cells. ROS plays a key and essential role in the modulation of normal physiological and biochemical processes occurring in cells1,2. The main source of ROS production in cells is from the mitochondrial electron transport chain (ETC) pathway as a part of the normal bioenergetic cycle. Significant additional sources of ROS production include enzymatic reactions such as cellular NADPH oxidases in cells. Metabolism of food molecules (e.g., glucose) occurs via the oxidative phosphorylation pathway in the mitochondrial matrix. A baseline level of ROS production is essential to regulate normal physiological cell signaling processes. Many key protein molecules that are part of the glucose metabolic signaling pathways (e.g., Akt and PTEN) are known to respond to intracellular ROS levels. Additionally, ROS are produced by various intracellular enzymes such as xanthine oxidase, nitric oxide synthase, and peroxisomal constituents as a part of the cellular enzymatic pathways1,2. In contrast to the natural sources of ROS, certain environmental factors, such as xenobiotics, infectious agents, UV light, pollution, cigarette smoking, and radiation, also lead to excessive production of ROS, which are a key driver of intra-cellular oxidative stress1,3. Elevated cellular oxidative stress can cause damage to native biomolecules inside a cell, such as lipids, proteins, and DNA, causing various diseases such as cancer, diabetes, cardiovascular disease, chronic inflammation, and neurodegenerative disorders1,3,4. Therefore, accurate measurements of ROS are essential to understand&#160;the cellular mechanisms involved in oxidative stress-induced disease pathophysiology.
Due to the short timescales of ROS production and elimination inside cells, quantitative measurements of various ROS radicals remains a challenge. Methods such as electron paramagnetic resonance (EPR)5, high-pressure liquid chromatography (HPLC), and fluorescence probe-based imaging are used to measure the various cellular ROS6. While methods such as EPR and HPLC yield quantitatively accurate estimates, these methods involve the destruction of the cellular spatial morphology and are usually in the form of global and bulk measurements of a sample. In contrast, imaging-based methods such as fluorescence probe-based methods retain the cellular morphology and spatial context of the ROS generation. However, the specificity of various fluorescence probes for different types of ROS radicals has not been well-established7,8. Several fluorescent probes such as dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (DCFH-DA), dihydrorhodamine (DHR), dimethyl anthracene (DMA), 2,7 dichlorodihydroflurescein (DCFH), 1,3-Diphenylisobenzofuran (DPBF), and MitoSox are available for ROS detection commercially. In the past decades, DHE, MitoSox, and DCFH-DA are the commonly used fluorescent dyes to measure ROS in cells and tissues8,9. DCFH-DA is a widely used dye for detecting intracellular H2O2 and oxidative stress. Despite the popularity of DCFH-DA, multiple previous studies have shown that it cannot be reliably used to measure intracellular H2O2 and other ROS levels8,9,10,11,12,13,14.
In contrast, the fluorescent probe dihydroethidium (DHE) shows a specific response to the intra-cellular superoxide radical (O2-). While the superoxide radical is one of many of the ROS species observed in cells, it is an important radical involved in the reduction of transition metals, conversion to peroxynitrate, and formation of hydroperoxides, among other intracellular effects. DHE is quickly taken up by the cells and has a fluorescence emission in the red wavelength range15. Upon reaction with superoxide radical specifically, DHE forms a red fluorescent product, 2-hydroxy ethidium (2-OH-E+). Thus, DHE may be considered as a specific probe for superoxide detection. However, DHE can also undergo nonspecific oxidation with ONOO- or OH., H2O2, and cytochrome c to form a second fluorescence product, ethidium E+, which can interfere with the measured 2-OH-E+ levels. However, these 2-OH E+ and E+ products, in combination, represent a major part of the total cellular ROS species observed inside a cell when stained with DHE. E+ intercalates into DNA, greatly enhancing its fluorescence8,9,10,11,13,14,15,16. Since the fluorescence spectra of ethidium and 2-hydroxy ethidium only differ slightly, the majority of ROS levels seen in a cell secondary to superoxide production can be detected and measured using DHE fluorescence products. These ROS species are identified using 480 nm wavelength excitation and 610 nm wavelength emission15,16,17.
In addition to choosing a specific fluorescent ROS detection probe, it is important to choose a sensitive method of detection to measure intracellular ROS. Accurate assessment of intracellular ROS levels is thus key to identifying disturbed redox balance states occurring in diseased cells or cells that have been exposed to various environmental stressors such as radiation, toxicological compounds, and genotoxic agents18. Since ROS is a commonly occurring phenomenon in cells that is responsible for regulating a variety of cell signaling activities, robust methods of detection of ROS are essential. To enable such high-throughput evaluation of ROS production within cells, this protocol uses a high-content screening (HCS) platform to measure the ROS species. The current protocol allows the high-throughput analysis of intracellular ROS production, which is of critical importance in many toxicology studies19. This protocol aims to provide an easy and versatile solution to detect and measure intracellular ROS in adherent hepatocellular carcinoma cells. The chemical reagents of H2O2 and menadione are used as potent ROS stimulators to measure the relative levels of ROS production in a controlled and high throughput setting. This protocol may be fine-tuned to measure ROS production in adherent and nonadherent cells under appropriate conditions, as necessary.

作者聚焦：肝细胞系中细胞内 ROS 水平的高通量测量

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

Our lab studies the impact of environmental pollutants, like heavy metals, on liver function and diseases related to reactive oxygen species production. This protocol aims to quantitatively measure ROS levels in cells using a dihydroethidium fluorescence probe. This protocol uses a high-content screening platform to quantitatively measure intracellular ROS species levels in hepatocellular lines.Measuring intercellular ROS level accurately is of critical importance in high-throughput toxicology study, which is a key area of interest in our lab. DHE fluorescence is a highly specific method for measuring total ROS levels, particularly targeting the superoxide radical, are giving it a significant advantage over other commonly used ROS fluorescence probe. Additionally, this protocol offer the unique ability to measure total cellular ROS level in a high-throughput platform.Our protocol is a robust and reproducible choice to characterize intracellular total ROS production. High-throughput approaches to measure ROS levels are helpful in a variety of studies, such as toxicology, drug screening, and cancer biology, which will be our focus in the future.

肝癌细胞的二氢乙铵 （DHE） 染色用于活性氧 （ROS） 的定量

首先，将 200 微升体积的 10， 000 个肝细胞癌细胞接种到 96 孔板中，加入 DMEM 和 10%FBS。将细胞在37摄氏度的加湿培养箱中孵育过夜，浓度为5%二氧化碳。第二天，更换培养基并用不同浓度的过氧化氢和甲萘醌处理细胞。将五毫克DHE溶解在一毫升DMSO中。对于工作溶液，用预热的DMEM培养基将100微摩尔等分试样稀释至10微摩尔浓度。将工作染料溶液涡旋 5 到 10 秒，以确保正确混合。现在，在静置癌细胞之前，从每个孔中取出测试培养基。然后用PBS轻轻清洗每个孔。将100微升工作染料溶液移液到每个孔中。将板在 37 摄氏度下孵育 30 分钟。从每个孔中取出含有DHE的培养基。使用多通道移液器用PBS清洗每个孔三次。现在，将 200 微升含有 Hoechst 33342 核染色染料的 PBS 移液到每个孔中。孵育后，将板转移到高内涵筛选平台。启动 Cellomics CX7 高内涵筛选软件。根据制造商的规格校准成像平台。在仪器数据库中打开特定于板品牌的系统参数。接下来，小心地将装有样品的板放在成像系统的加热台上。确保板位置牢固且方向正确。然后轻轻按压微孔板，确保其平放在载物台上。板正确定位后，按住 Control 键。然后单击“控制”（Control） 和“确定”（OK） 以打开“板加载/卸载”（Plate Load/Unload） 对话框。要选择成像参数，请在 Cellomics BioApplications 界面中打开靶标激活模式。创建与核染色和DHE荧光探针兼容的双通道数据采集新方案。接下来，指定图像采集所需的物镜，然后选择合适的放大倍率。现在指定曝光时间、相机弯曲和 z 堆叠间隔。设置成像参数后，使用仪器中的不同算法识别感兴趣的主要跟踪对象。分割已识别的物体后，根据每种细胞类型特有的特定大小、形状和强度参数验证主要物体。定义对象周围的感兴趣区域，并在其周围设置一个 20 微米的圆圈。单击“群体表征”选项卡，然后将扫描限制设置为每孔 1000 个细胞，每个字段至少 10 个对象。现在单击“启动视图”以扫描每个孔板。然后启动视图分析软件，以CSV格式导出定量数据参数，以进行其他分析。过氧化物暴露导致所有细胞系的ROS诱导荧光以剂量依赖性方式在统计学上显着增加。使用甲萘醌时，JHH-4细胞的荧光强度呈剂量依赖性增加。然而，仅在其他两种细胞系中观察到较高浓度的荧光差异。

Research

JoVE Journal

Bioengineering

Dihydroethidium &#40;DHE&#41; Staining of Hepatocarcinoma Cells for the Quantification of Reactive Oxygen Species &#40;ROS&#41;

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 ...

科研

生物工程

肝癌细胞的二氢乙铵 （DHE） 染色用于活性氧 （ROS） 的定量

肝癌细胞的二氢乙铵（DHE）染色用于活性氧（ROS）的定量