우리는 이러한 연구들의 관대한 지원 ReadiSorb 제품 감사합니다.

1. 세포 고양이 astrocytes의 문화와 H 2 O 2와 치료 <ol><li> 37 DMEM 미디어 플러스 영양소와 75cm이 플라스크에 문화 세포주 G355 - 5 ° C와 5% CO 2 60 % 합류 (~ 5 일)까지. 모든 1~2일 미디어를 교체 또는 건강한 문화를 유지하는 데 필요한. </li><li> 미디어를 제거하고 0.25 %의 트립신 2 ML를 추가합니다. 45 S에 대한 pipetting하여 세포를 리프트 - 실내 온도 (RT)에서 1 분. </li><li> 신속 미디어 10 ML을 포함한 15 ML 원뿔 관에 현탁액을 전송하기만하면됩니다. 부드럽게 섞는다. </li><li> 펠렛을 얻기 위해 3 분 300 XG, 23 ° C에서 원심 분리기. 펠렛을 방해하지 않고 뜨는 폐기하십시오. </li><li> 신선한 미디어 6-7 ML을 추가하고 세포를 resuspend. </li><li> 6 잘 플레이트의 각 잘하는 셀 ~ 1 ML을 추가합니다. 각 물론 미디어로부터 2-3 ML을 추가하고 80~90%의 합류까지 품어. </li><li> 미디어 과산화수소의 100μM 솔루션 (H 2 O 2)를 준비합니다. </li><li> 각 우물에서 미디어를 제거하고 하나 둘 ML 신선한 매체 (제어), H 2 O 2 (치료) 또는 DPBS의 2ml 2 ML로 바꿉니다. ° C, 5 % CO 2 3 H. 위해 37 알을 품다 </li><li> 솔루션을 제거하고 세포를 리프트하는 트립신 1 ML를 추가합니다. 0.5 ML의 DPBS을 포함하는 1.7 ML Eppendorf 튜브에 세포를 전송합니다. 이 단계는 연장 트립신 보육에서 세포 죽음을 방지하기 위해 각 잘 개별적에서 수행되어야합니다. </li><li> 3 분 300 XG에 스핀, 버리고 뜨는 및 DPBS 1 ML에 resuspend. </li></ol> 2. ROS를위한 염색법 <ol><li> 얼룩의 모든 단계 표백 방지하기 위해 최소한의 빛의 노출을 실시해야합니다. </li><li> 긍정적인 제어 50 MM의 CCCP 1 μl를 추가합니다. 37 품어 ° C, 5 % 5 분 CO 2. </li><li> H 2 O 2 그룹 10 MM CM - H 2 DCFDA 솔루션 5 μl를 추가합니다. 37 품어 ° C 5 % CO 15-30 분 2. 아무 얼룩은 생리 예제에 추가하지 않습니다. </li><li> 잔여 얼룩을 제거하는 DPBS 2 ML과 샘플을 씻으십시오. </li><li> 펠렛을 얻기 위해 3 분 300 XG에 스핀. </li><li> DPBS 0.5 ML에서 세포를 Resuspend. 생리를 제외하고, 각 예제 1 μl PI를 추가합니다. </li><li> 5ml 흐름 cytometer 튜브에 샘플을 전송합니다. </li><li> 흐름 cytometer를 실행합니다. </li></ol> 3. 유동세포계측법 <ol><li> 525, 775 및 620 nm의 모든 ± 20 nm의 : 488 nm의 아르곤 레이저 다음과 같은 밴드 패스 갖춘 베크맨 쿨터 FC500 (또는 이에 상응하는 금액)에서 샘플을 실행합니다. </li><li> 해당 컨트롤을 준비 : 흠없는 세포가, CM - H 2 DCFDA은 (대조군) PI 단, 더블 스테인드 밖에 스테인드. </li><li> FS 대 SS, PI 대 CM - H 2 DCFDA 다음 histoplots을 가진 프로토콜을 확인합니다. 또한 다음과 같은 histograms을 가집니다 얼룩에 해당하는 휴대폰 번호 대 FL1, FL2 및 FL3을. </li><li> 샘플을 실행하기 전에, 장비, 유체 및 폐기물 컨테이너를 확인합니다. 최소한 20 분 장비를 따뜻하게. </li><li> 장비를 청소하고 제조 업체의 표준에 따라 적절한 주판으로 보정을 수행합니다. </li><li> 귀하의 프로토콜을 선택하고 샘플을 실행합니다. </li><li> 이전 완료로 장비를 청소하십시오. </li><li> 제조 업체의 기준에 따라 진공 라인을 청소하십시오. </li><li> 흐름을 끄고 머리 / 진공 라인을 청소하십시오. </li><li> 소프트웨어 및 컴퓨터를 끕니다. </li></ol> 4. 대표 결과 : 유동세포계측법 결과는 FlowJo 7.6과 얼룩 컨트롤이 객관적으로 게이팅을 설정하는 데 사용되었다를 사용하여 분석했다. 이것을 근거로, 건강한 세포가 히스토그램 및 ROS (산화 스트레스)의 왼쪽에 표시가 오른쪽으로 세포의 변화로 감지되었다. 그림 1은 건강한 고양이 astrocytes에 과산화수소의 효과의 결과를 보여줍니다. FL1에서 데이터는 ROS의 존재를 나타내는 CM - H 2 DCFDA의 강도를 측정하는 데 사용되었다. 예상했던대로, ROS의 높은 금액은 H 2 O 2 대우 샘플에서 발견되었다. 이것은 왼쪽에서 오른쪽으로 형광 강도의 변화로 히스토그램에 표시됩니다. DMEM 대 DPBS 취급 건강한 세포를 비교하면, ROS의 양을 (그림 2)에는 큰 변화가 없었다. <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="그림 1"/> 건강한 고양이 astrocytes에서 산화 스트레스의 수준과 건강한 세포에 H 2 O 2의 효과 그림 1. 유동세포계측법 결과. 결과없이 치료 (DMEM)과 세포와 비교하여 H 2 O 2 건강한 세포에 ROS의 수준을 증가시킬 수있다는 것을 나타냅니다. <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="그림 2"/> DPBS와 3hrs에 대한 incubated 건강한 세포에 감지 ROS의 수준의 그림 2. 유동세포계측법 결과.

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바이러스 자주 제안된 메커니즘의 연결 손상이 산화 스트레스 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31 유도. 기본적으로, 그것은 제안 이러한 수산화, 초과 산화물의 음이온, 질산 산화물 및 / 또는 신경에​​ 독성이 과산화수소, 같은 바이러스성 노출을 통해 glia (astrocytes와 microglia)가 릴리스 반응 산소 래디 칼. 마찬가지로, 산화 스트레스는 또한 암페타민 유발 neurotoxicity 2, 6, 17의 주요 pathologic 메커니즘으로 제안되었습니다. 우리 그룹 CNS에 대한 학대의 바이러스와 약물 모두의 영향에 관심이 있기 때문에, 우리는 astrocytes에 초과 ROS의 현재를 결정하는 빠른 선별 검사를 사용하여 선출. 의 연결을 글루타티온의 대부분은 astrocytes 제작한 다음 뉴런 5, 8, 10-12, 24, 26, 29, 30로 전달됩니다. 따라서, 산화 스트레스에 astrocytes 역할은 뉴런에 생체 항상성 환경을 유지뿐만 아니라 ROS에서 뉴런을 보호하는 중요한이며, astrocyte 세포 배양이 연구를 위해 선택한 것을 이유이다. CM - H 2 DCFDA도 2 &#39;, 7&#39; - dichlorofluorescein 및 H2DCF로 알려져 있습니다. CM - H 2 DCFDA는 반응 산소 종의 존재에 대한 세포 침투성 표시기입니다. CM - H 2 DCFDA 세포 esterases은 아세테이트 그룹을 제거한 후 형광 될 아닌 형광 화합물로 세포를 입력하고, 화합물이 산화된다. 일단 세포와 아세테이트 그룹 내부에 제거됩니다, CM - H 2 DCFDA는 녹색으로 결과, 질산 산화, peroxynitrite의 anions, 과산화수소, 그리고 유기 hydroperoxides 3, 14, 25를 포함하여, ROS 종의 숫자에 의해 산화 수 있습니다 형광 제품 23, 28. 이러한 부족 CM - H 2 DCFDA을 산화 수있는, ROS에 지정한 산화 스트레스 메커니즘이 역할을로 여겨지고있는 pathogenesis 조사의 초기 단계에서 가치 시약 만들어지만, 어떤 알 수있다 급진적인 산소 오히려 각 ROS 별도의 시험보다 참여 수 있습니다. 일단 그것은 ROS가 ROS 또는 로스의 조합이 특정 pathogenesis 과정에 관련된 CM - H 2 DCFDA 다음, 추가 실험을 확인하기 위해 수행할 수의 산화에 의해 존재하는 것을 설립되었습니다. 생리 조절에 비해이 현재 연구의 결과는 나타내는 과산화수소 CM - H 2 DCFDA 증가의 추가와 함께 발견되었다 형광 것을 보여주는 이번 분석은 G355 - 5 이내에 산화 환경을 검출 가치 테스트입니다 전지, 고양이 astrocyte 세포 라인.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013 - CV </td><td></td></tr><tr><td> 멤 비타민 </td><td> Cellgro </td><td> 25-020 - CL </td><td> 1퍼센트 (V / V) </td></tr><tr><td> L - 글루타민 </td><td> Hyclone </td><td> 17 - 605E </td><td> 1퍼센트 (V / V) </td></tr><tr><td> 비 필수 아미노산 </td><td> Hyclone </td><td> 25-025 - CL </td><td> 1퍼센트 (V / V) </td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td> 20% (V / V) </td></tr><tr><td> 필수 아미노산 </td><td> Cellgro </td><td> 25-030 - CL </td><td> 0.2 % (V / V) </td></tr><tr><td> 500ml 진공 여과 시스템 </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ML 원뿔 튜브 </td><td> 매 </td><td> 352097 </td><td></td></tr><tr><td> 75cm 두 조직 문화 flasks </td><td> 매 </td><td> 353136 </td><td></td></tr><tr><td> 6 잘 조직 배양 플레이트 </td><td> 매 </td><td> 353224 </td><td></td></tr><tr><td> CM - H 2 DCFDA </td><td> Invitrogen </td><td> C6827 </td><td></td></tr><tr><td> Propidium의 이우 (PI) </td><td> 시그마 </td><td> P4170 - 10mg </td><td></td></tr><tr><td> 트립신 </td><td> Lonza </td><td> 17 - 160F </td><td></td></tr><tr><td> H 2 O 2 </td><td> 시그마 </td><td> H6520 </td><td></td></tr><tr><td> HyQ의 항생제 </td><td> Hyclone </td><td> SV30079.01 </td><td> 0.1 % (V / V) </td></tr><tr><td> G355 - 5 전지 </td><td> ATCC </td><td> CRL - 2033 </td><td> 일반 고양이 뇌 </td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

의 연결을 손상을 유도 바이러스의 자주 제안 메커니즘은 산화 스트레스입니다. Astrocytes는 중추 신경계 (CNS)의 산화 스트레스를 조절에 중요​​한 역할을했습니다. Astrocytes는 반응 산소 종 (ROS)에서 뉴런뿐만 아니라 보호하는 뉴런에 생체 항상성 환경을 유지할 수 있도록 도와주십시오. CM - H2DCFDA는 ROS의 존재에 대한 세포 침투성 표시기입니다. CM - H 2 DCFDA이 아닌 형광 화합물로 세포를 입력하고, 휴대폰 esterases은 아세테이트 그룹을 제거한 후 형광되고, 화합물이 산화됩니다. 녹색 fluorescing 것으로 판단될 유동세포계측법에 의해 측정한 세포의 숫자는 산화 상태에있는 세포의 수를 나타냅니다. CM - H 2 DCFDA 다른 ROS 다수에 의해 산화 쉽습니다. 이 분석에 관계없이하는 산소의 산화 세포 환경을 화면으로 사용할 수로 ROS는 CM - H 2 DCFDA을 산화 수있는에 대한 특이성의이 부족은,이 화합물 pathogenesis 조사의 초기 단계에 사용하기 위해 가치있는 리젠트합니다 ROS의 급진 또는 조합은 세포 조건에 책임이 있습니다. 일단 그것은 ROS가 CM - H 2 DCFDA의 산화에 의해 현재 다음 추가 실험이 ROS 또는 로스의 조합이 특정 pathogenesis 과정에 참여하는 결정하기 위해 수행할 수있는 것으로 설립되었습니다. 본 연구의 결과는 과산화수소 CM - H 2 DCFDA의 증가 또한이 분석은 G355 - 5 세포 내에서 산화 환경을 검출을위한 가치있는 테스트입니다 나타내는 생리 컨트롤에 상대적으로 감지되었습니다 형광 것을 입증 고양이 astrocyte 세포 라인.

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

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

산화 세포 환경을 감지하는 심사 방법은 CM - H2DCFDA의 산화를 측정하는 것입니다. 일단 비 형광의 형광 화합물로 세포, CM - H2DCFDA 변경 내에 산화. 형광에이 변경 사항은 유동세포계측법으로 측정하고 산화 환경에서 세포의 수를 나타냅니다.

고양이 Astrocyte 세포주에서 산화 스트레스에 대한 심사 분석, G355 - 5

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

screening-assay-for-oxidative-stress-feline-astrocyte-cell-line-g355

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.4K 조회수.  Western University of Health Sciences. 산화 세포 환경을 감지하는 심사 방법은 CM - H2DCFDA의 산화를 측정하는 것입니다. 일단 비 형광의 형광 화합물로 세포, CM - H2DCFDA 변경 내에 산화. 형광에이 변경 사항은 유동세포계측법으로 측정하고 산화 환경에서 세포의 수를 나타냅니다.

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

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.

유전자로 인코딩된 Fluorogenic 센서를 사용 하 여 독성 산화 스트레스의 평가에 접근을 이미징

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

연구

암 연구학

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

생물학

GSH/GSSG 측정 – 항산화 연구를 위한 정제된 OPA 분석

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

저자 스포트라이트: 산화 스트레스 연구를 위한 혁신적인 티올 정량화 및 바이오마커 검출 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

In vitro에서 배양된 포유류 세포에서 ortho -phthaldehyde를 사용한 산화 및 환원 형태의 글루타치온의 신속한 정량화

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

생화학

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

사이토 반응성 및 확산에서 체 외에 허 혈 성 같은 조건 모니터링

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

신경과학

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

문화의 세포에 대한 생존 분석 실험

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

간암 세포에서 Dihydroethidium을 사용한 새로운 ROS 정량 분석

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.

저자 스포트라이트: 간세포주에서 세포 내 ROS 수준의 고처리량 측정

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.

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

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

고양이 Astrocyte 세포주에서 산화 스트레스에 대한 심사 분석, G355 - 5

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In vitro에서 배양된 포유류 세포에서 ortho -phthaldehyde를 사용한 산화 및 환원 형태의 글루타치온의 신속한 정량화

In vitro에서 배양된 포유류 세포에서 ortho -phthaldehyde를 사용한 산화 및 환원 형태의 글루타치온의 신속한 정량화

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문화의 세포에 대한 생존 분석 실험

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

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

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