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

간암 세포에서 Dihydroethidium을 사용한 새로운 ROS 정량 분석

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.

저자 스포트라이트: 간세포주에서 세포 내 ROS 수준의 고처리량 측정

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

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.

활성산소종(ROS)의 정량화를 위한 간암 세포의 디하이드로에티듐(DHE) 염색

시작하기 위하여는, DMEM와 10%FBS를 가진 96의 좋은 판으로 10의 000의 간세포 암종 세포의 200 마이크로리터 양을 씨를 뿌립니다. 이산화탄소 5% 미만의 가습 인큐베이터에서 섭씨 37도에서 밤새 세포를 배양합니다. 다음날 배양 배지를 교체하고 세포를 다양한 농도의 과산화수소와 메나디온으로 처리합니다.5mg의 DHE를 DMSO 1밀리리터에 녹입니다. 작업 용액의 경우, 100마이크로몰 부분 표본을 예열된 DMEM 배지로 10마이크로몰 농도로 희석합니다. 적절한 혼합을 보장하기 위해 5-10초 동안 작업 염료 용액을 소용돌이치십시오.이제 암종 세포를 세우기 전에 각 웰에서 테스트 배지를 제거합니다. 그런 다음 PBS로 각각을 부드럽게 잘 씻으십시오. 각 웰에 100 마이크로 리터의 작업 염료 용액을 피펫팅합니다.접시를 섭씨 37도에서 30분 동안 배양합니다. 각 웰에서 DHE가 포함된 매체를 제거합니다. 다중 채널 피펫을 사용하여 PBS로 각 웰을 세 번 세척합니다.이제 Hoechst 33342 핵 염색 염료가 포함된 PBS 200마이크로리터를 각 웰에 피펫팅합니다. 배양 후 플레이트를 high-content screening 플랫폼으로 옮깁니다. Cellomics CX7 High-Content 스크리닝 소프트웨어를 실행합니다.제조업체의 사양에 따라 이미징 플랫폼을 보정합니다. 기기 데이터베이스에서 플레이트 브랜드와 관련된 시스템 파라미터를 엽니다. 다음으로, 샘플이 있는 플레이트를 이미징 시스템의 가열된 스테이지에 조심스럽게 놓습니다.플레이트가 단단히 배치되고 올바른 방향인지 확인하십시오. 그런 다음 마이크로플레이트를 부드럽게 눌러 s에 평평하게 놓이도록 합니다.tag이자형. 플레이트가 올바르게 배치되면 Control 키를 길게 누릅니다.그런 다음 Control(제어) 및 OK(확인)를 클릭하여 Plate Load/Unload 대화 상자를 엽니다. 이미징 파라미터를 선택하려면 Cellomics BioApplications 인터페이스에서 Target Activation 모드를 엽니다. 핵 염색 및 DHE 형광 프로브와 호환되는 듀얼 채널 데이터 수집을 위한 새로운 프로토콜을 생성합니다.그런 다음 이미지 획득에 필요한 대물렌즈를 지정한 다음 적절한 배율을 선택합니다. 이제 노출 시간, 카메라 벤딩 및 Z 스택 간격을 지정합니다. 이미징 파라미터가 설정되면 기기의 다양한 알고리즘을 사용하여 관심 있는 기본 추적 개체를 식별합니다.식별된 개체를 분할한 후 각 세포 유형에 고유한 특정 크기, 모양 및 강도 매개변수를 기반으로 기본 개체를 검증합니다. 개체 주변의 관심 영역을 정의하고 그 주위에 20마이크로미터 원을 설정합니다. Population Characterization(모집단 특성화) 탭을 클릭한 다음 스캔 제한을 웰당 1000개 셀로 설정하고 필드당 최소 10개의 개체를 설정합니다.이제 Launch View(보기 시작)를 클릭하여 각 웰 플레이트를 스캔합니다. 그런 다음 뷰 분석 소프트웨어를 실행하고 추가 분석을 위해 정량적 데이터 매개변수를 CSV 형식으로 내보냅니다. 과산화물 노출은 용량 의존적 방식으로 모든 세포주에 걸쳐 ROS 유도 형광의 통계적으로 유의한 증가를 초래했습니다.menadione로, JHH-4 세포는 형광성 강렬에 있는 복용량 의존하는 증가를 보여주었습니다. 그러나 형광의 현저한 차이는 다른 두 세포주에서 더 높은 농도에서만 관찰되었습니다.

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 ...

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