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.

<ol>
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저자는 경쟁하는 금전적 이해 관계 나 다른 이해 상충이 없다고 말합니다. 또한 저자는 모든 저자가 모든 이해 상충을 공개하도록 요청 받았습니다.

이 논문은 NHDF에서 세포 내 ROS 수준과 미토콘드리아 morphofunction의 동시 정량화를위한 ​​고 함량 현미경 검사법을 설명합니다. 그 성능은 SQV가 처리 된 NHDF에 대한 사례 연구를 통해 입증되었습니다. 그 결과는 별도의 실험 19 , 20 , 21 , 22 , 23 , 24 <su...

<html> 세포 내 ROS의 농도는 ROS 생성 시스템과 ROS를 제거하는 시스템 사이의 동적 상호 작용을 통해 세 심하게 조절됩니다. 이 둘의 불균형은 산화 스트레스 상태를 일으킨다. ROS의 주요 원인 중 하나는 미토콘드리아이다. 세포 호흡에서의 역할을 감안할 때, 그들은 세포 내 superoxide (O 2 • - ) 분자의 대부분을 담당합니다 2 . 이것은 주로 강한 마이너스 내부 미토콘드리아 막 잠재력 (Δψ m ), 즉 미토콘드리아 과분극 조건 하에서 전자 전달 사슬의 복합체 1에서 O 2 로의 전자 누출에 기인한다. 반면에 mitochondrial depolarization은 ROS 생성 증가와 상호 작용하여 여러 형태의 작용 3 , 4 , 5 ,&gt; 6 , 7 , 8 . 또한 핵분열 - 핵융합 기계 단백질의 산화 환원 변형을 통해 ROS는 미토콘드리아 형태를 공동 조절한다. 예를 들어, 단편화는 ROS 생성 및 세포 사멸 증가와 관련이있다 10 , 11 , 섬유 성 미토콘드리아는 영양 기아와 보호 mitophagy 12 . 세포 ROS와 미토콘드리아 morphofunction 사이의 복잡한 관계를 감안할 때, 둘 다 살아있는 세포에서 동시에 정량화되어야합니다. 정확하게 이것을 수행하기 위해, 형광 프로브 CM-H 2 DCFDA (ROS) 및 TMRM (미토콘드리아 Δψm 및 형태학)으로 염색 된 부착 세포 배양 물의 자동화 된 와이드 필드 현미경 및 이미지 분석에 기초하여 고 - 컨텐츠 이미징 분석법을 개발 하였다. 고 - 컨텐츠 이미징은 sp다중 보완 마커 및 자동화 된 이미지 분석을 사용하여 세포 phenotypes에 대해 atiotemporally 풍부한 ( 즉 , 많은 설명 기능) 정보. 자동화 된 현미경 검사와 결합하면 많은 샘플을 병렬로 ( 즉 높은 처리량) 스크리닝 할 수 있으므로 분석의 통계 능력이 향상됩니다. 사실, 프로토콜의 주요 자산은 같은 세포에서 여러 매개 변수의 동시 부량을 허용하고, 이것은 많은 세포 및 조건에서 가능하다는 것입니다. 프로토콜은 8 부분으로 나누어 져 있습니다 (아래의 프로토콜에 자세히 설명되어 있습니다) : 1) 96- 웰 플레이트에서 세포를 시드; 2) 원액, 작업 용액 및 영상 버퍼의 준비; 3) 현미경 셋업; 4) CM-H 2 DCFDA 및 TMRM으로 세포를 로딩하는 단계; 5) 기본 ROS 수준과 미토콘드리아 morphofunction을 측정하는 최초의 라이브 영상 라운드; 6) tert- 부틸의 첨가 후 둥근 두번째 이미지 생성유도 된 ROS 수준을 측정하기위한 과산화수소 (TBHP); 7) 자동화 된 이미지 분석; 8) 데이터 분석, 품질 관리 및 시각화. 이 분석법은 원래 정상적인 사람 피부 섬유 아세포 (NHDF)를 위해 개발되었습니다. 이러한 세포는 크고 평평하기 때문에 2D 와이드 이미지 13 , 14 에서 미토콘드리아 형태학을 평가하는 데 적합합니다. 그러나 사소한 수정으로이 방법은 다른 접착 세포 유형에도 적용 할 수 있습니다. 또한, CM-H 2 DCFDA와 TMRM의 조합 옆에 워크 플로우는 다른 분자 특이성 1 , 15 의 다양한 형광 염료 쌍을 준수합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>Reagents</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 &#181;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 1M 500mL</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&#39;)&#8322; 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&#39;)&#8322; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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

활성 산소 종 (ROS)은 유전자 발현, 이동, 분화 및 증식을 포함한 필수 세포 과정을 조절합니다. 그러나 과도한 ROS 수준은 DNA, 지질 및 단백질에 대한 비가 역적 산화 손상을 수반하는 산화 스트레스 상태를 유도합니다. 따라서, ROS의 정량화는 세포 건강 상태에 대한 직접적인 프록시를 제공한다. 미토콘드리아가 ROS의 중요한 세포 원 및 표적이기 때문에 같은 세포에서 미토콘드리아 기능과 ROS 생산의 공동 분석은 병리 생리 학적 조건에서 상호 연결을 더 잘 이해하는 데 중요합니다. 따라서, 세포 내 ROS 수준, 미토콘드리아 막 잠재력 (ΔΨ m ) 및 미토콘드리아 형태학의 동시 정량화를위한 ​​고 함량 현미경 기반 전략이 개발되었습니다. 그것은 다중 웰 플레이트에서 성장한 살아있는 부착 세포의 자동화 된 광역 형광 현미경 및 이미지 분석에 기반을두고 있습니다.세포 - 투과성 형광 리포터 분자 CM-H2 DCFDA (ROS) 및 TMRM (ΔΨm 및 미토콘드리아 형태학)을 사용하여 d를 측정 하였다. 형광 측정 또는 유동 세포 계측법과는 달리,이 전략은 실험 자극 전후 모두 높은 시공간 해상도를 갖는 개별 세포 수준에서 세포 매개 변수를 정량화 할 수 있습니다. 중요하게도,이 방법의 이미지 기반 성질은 신호 강도 이외에 형태 론적 파라미터를 추출 할 수있게한다. 결합 된 기능 세트는 하위 집단, 세포 유형 및 / 또는 치료법 간의 차이를 탐지하기위한 탐색 및 통계 다변량 데이터 분석에 사용됩니다. 여기에, 화학 섭동 후 세포 상태 사이의 모호하지 않은 차별을위한 잠재력을 증명하는 실험 예와 함께 분석에 대한 자세한 설명이 제공됩니다.

분석은 여러 대조 실험을 사용하여 벤치마킹되었으며, 그 결과는 Sieprath et al. 1 . 간단히 말하면, CM-H 2 DCFDA 및 TMRM의 세포 내 ROS 및 Δψ m의 외적으로 유도 된 변화에 대한 형광 응답을 각각 동적 범위를 결정하기 위해 정량화 하였다. CM-H 2 DCFDA의 경우, NHDF는 10 μM에서 160 μM 범위의 TBHP 농도로 처리했을 때 형광 신호의 선형 ?...

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

Cellular Redox Profiling Using High-content Microscopy

이 논문은 세포 내 ROS 수준과 미토콘드리아 막 전위와 형태학을 동시에 정량화하기위한 고 - 내용 현미경 검사 워크 플로우를 제시한다. 세포 투과성 형광 리포터 분자 5- (and- 6) - 클로로 메틸 -2 &#39;, 7&#39;- 디클로로 디 히드로 플루 오렌 시스 디 아세테이트, 아세틸 에스테르 (CM-H 2 DCFDA) 및 테트라 메틸 로다 민 메틸 에스테르 (TMRM)

고감도 현미경을 이용한 세포 산화 환원 프로파일 링

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

10.8K 조회수.  University of Antwerp.  이 논문은 세포 내 ROS 수준과 미토콘드리아 막 전위와 형태학을 동시에 정량화하기위한 고 - 내용 현미경 검사 워크 플로우를 제시한다. 세포 투과성 형광 리포터 분자 5- (and- 6) - 클로로 메틸 -2 ', 7'- 디클로로 디 히드로 플루 오렌 시스 디 아세테이트, 아세틸 에스테르 (CM-H 2 DCFDA) 및 테트라 메틸 로다 민 메틸 에스테르 (TMRM) 

비디오: Cellular Redox Profiling Using High-content Microscopy

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 ...

연구

생물학

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF 현미경과 망막 바이폴라 세포 이미징 Exocytosis

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

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.

동위 원소 태그 질량 분광법을 사용하여 Thiol 산화 환원 프로테옴 프로 파일링

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

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

표현 복제가 Grb2 신호 복합 단지를 공부하기 위해 높은 콘텐츠 이미징 워크 플로우

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

탠덤 고압 냉동 및 투과 전자 현미경을위한 식물 조직의 빠른 동결 교체

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

자동 형광 수명 이미징 현미경을 활용하여 오픈 소스 높은 콘텐츠 분석

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.