우리는 기꺼이 우리의 연구실과 기술 지원 및 도움 토론 고령화와 영양 (CISBAN)의 통합 시스템 생물학을위한 센터의 모든 구성원을 인정합니다. 이 연구는 생명 공학 및 생물 과학 연구 협의회 (BBSRC) (BB/C008200/1)와 Wellcome 트러스트 (075,294, 093,088)에 의해 지원되었다.

<ol>
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관심의 어떠한 충돌 선언 없습니다.

QFA은 여러 감각에 직접 셋 설립 벤치 규모의 미생물 기술의 후손 : 문화 희석 시리즈 스포트 테스트 9 aerated 액체 문화 9, 복제 도금 13 성장 곡선 결정. 이러한 세 가지 방법을 요약하고 QFA 및 표 2에있는 다른 높은 처리량 기법과 비교됩니다. 희석 시리즈 스폿 테스트 inoculum의 dilutions의 범위에서 성장 문화 일련의 식​​민지를 형성하는 기능으로 변형의 적합성을 정의하는 반면, 단일 문화의 반복 관측에 의한 QFA 측정 스트레인 헬스는 성장 곡선을 만들 수 있습니다. 단일 문화권에서 체력을 Quantifying 것은 더 많은 변종이 동일한 조건 하에서, 동시에 검사 할 수 있습니다. 다른 환경이나 유전적 배경에있는 문화 운동의 차이에 대해 테스트할 때 문화의 복제 배열은 대부분의 유용 스트레인 컬렉션의 반복 테스트를하실 수 있습니다. QFA 프로토콜 제공여기에, 독립적인 문화 (로봇 QFA, 표 2) 수천의 성장을 관찰 적합한 부화 및 이미지 한천 플레이트를 복제 예방하기 위해 고가의 로봇 장비를 사용합니다. QFA가 설립, 전통적인 실험실 기법을 기초로했기 때문에하지만, 그것은 또한 수동 단계 (수동 QFA, 표 2)와 로봇의 도움을 대체하여보다 싸게 많이 수행 할 수 있습니다. 수동 QFA는 수동 핀 도구 및 및 사진을위한 인큐베이터에서 접시의 수동 전송을 사용하여 한천 플레이트를 향해 문화 복제 및 접종을 포함한다. 동일한 전산 분석 워크플로우는 두 실험적인 디자인의 성장 곡선을 생성하는 데 적용할 수 있습니다. QFA 휘트니스 견적은 비교적 정확하게 될 것 같습니다. 그림 4에서 기능적으로 관련 유전자 삭제 네 가지 예제 클러스터의 평균 fitnesses가 강조 표시됩니다. 이 independ의 각 기능과 관련된 유전자 클래스의 멤버 근접엔트 유전적 배경 (ura3Δ 및 cdc13-1) QFA 휘트니스 견적의 재현성을 나타냅니다. 예를 들어, 불과 3 유전자 삭제는 (아웃 가능 4,300의) 보존 MRX 복합의 세 멤버를 구분합니다. 미생물 종자의 성장 특성을 테스트하기 위해 QFA까지 고속 처리 대안 SGA 5,6, 분광 플레이트 판독기에 광학 밀도 속도론을 캡처 경쟁 barcoded 라이브러리에서 화면 11,12와 스크린을 포함 표 2에 요약하며 더욱 완벽하게 다른 여덟 설명되어 있습니다 10 . QFA와 문화가 고체 한천 표면 (한천 기반 방법, 표 2)에서 자라는 SGA 판은 로봇에 의해 신속하고 간편하게 처리할 수 있습니다. 고체 한천 표면 문화가 잘 성장 전역 aerated하고 전지는 사교적인 미생물들이 지역 사회 14 성장할 수 있도록 고정 문화권으로 성장할 수있다. 왼쪽 그대로, 미생물 커뮤니티자신의 마이크로 환경 등 에탄올과 같은 secreting 독소, 그리고 아마도 세포 사이에 신호를 ffect. 그러나 적절한 통기를 달성하는 데 필요한 액체 문화의 혼합 지속적인 성장을 인위적으로 자신의 모드에 영향을 미칠 수있는 미생물 지역 사회와 그들의 마이크로 환경을 방해. 문화 사이의 세포를 나르는 액체 많아요을위한 더 적은 기회가 있기 때문에 교차 오염은 고체 한천 방법에 우려의 적습니다. 오염 고체 한천 assays 외국 공기 매개로 미생물에 의해 발생하는 경우, 그것은 흔히 고체 한천 플레이트의 육안 검사에 의해 감지 집결하거나 제거할 수 있습니다. QFA 처리량은 두 가지 방법으로 병렬 액체 방법에 비해 훨씬 높다. 우선, QFA (그리고 SGA) 접시에 주사 문화는 접시 당 독립적인 문화를 제공,보다 밀도가 함께 포장됩니다. QFA 년 308 문화권이 아닌 실험적인 첨단 문화 (그림 1) co.kr 사이트를 세지 않고, 일반적으로있다판 당 96 또는 100의 문화와 액체 문화를 병렬하는 앞에 서. 둘째, QFA 실험은 접시의 훨씬 더 큰 숫자에 따라 결정됩니다. 단일 QFA 실험에서 분석 번호판의 숫자에만 보육이나 따뜻한 방, 최소 허용 이미지 캡처 주파수와 최대 달성 캡처 주파수에서 사용 가능한 공간에 의해 제한되는 반면, 액체 성장 실험 접시의 수를 강력히 제한 플레이트 리더 (일반적으로 하나 또는 두 개의 접시) 또는 플레이트 리더 (일반적으로 25-50 접시)에 연결된 스태커의 용량에 의해. 우리는 최근에 37,884 동시 문화를주는 308 실험적인 문화를 가진 123 접시, 각에서 완전 자동화된 QFA 실험을 실시했습니다. 우리는 액체로 성장하고 독립적인 문화의 최대 달성 번호 96 (문화 / 플레이트)는 것을 예상하고 우리 QFA 처리량보다 열 배 가량 X 50 (플레이트 / 스태커) = 4,800. 액체 배양 화면에 대한 대안은 많은 자동 촉진제을 활용하는 것입니다병렬 wth 장치 (Blomberg 8 시까지 검토에서 표 1), 하나 또는 두 개의 접시의 용량을 가진 각. 각 장치의 온도 제어가 독립적이고, 그래서 조건이 똑같은 건 아니지만, 그들이한다고 가정하고,이 흐름은 적어도 190과 같은 장치가 QFA 처리량 (장치 당 200 액체 문화를 가정)과 일치하도록 요구한다. 요약에서는 QFA는 유용 소규모 focussed 실험 및 높은 처리량 화면에 모두 양적 성장 phenotypes를 수집하기 위해 적용할 수있는 고품질의 흐름이다. 그것은 로봇 장비에 대한 요구 사항 변화에 효과적으로 적용될 수있을만큼 유연합니다. QFA 작업 흐름의 전산 구성 요소를 자유롭게 사용할 오픈 소스 코드를 기반으로합니다.

<table border="1"><tbody><tr><td> 시약 / 장비의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 복제 광택히 </td><td> 시그마 </td><td> R-2508 </td><td> 1 / 8 &quot;직경 핀으로 96 핀 설명서 pintool </td></tr><tr><td> 메이트를 혼용 </td><td> Eppendorf </td><td> 5353 000.014 </td><td></td></tr><tr><td> Biomek FX </td><td> Beckman </td><td> A31842 </td><td></td></tr><tr><td> Teleshake </td><td> 써모 과학 </td><td> 50095890 </td><td> Biomek FX에 설치된 </td></tr><tr><td> BM3-SC </td><td> S &amp; P 로보틱스 INC </td><td> BM3-SC </td><td> 192 선반 회전 회전 목마 </td></tr><tr><td> spImager </td><td> S &amp; P 로보틱스 INC </td><td> spImager </td><td> 고해상도 수동 이미징 </td></tr><tr><td> Cytom있는 spImager에 </td><td> S &amp; P 로보틱스 INC </td><td> 사용자 정의 </td><td> 온도는 자동 고해상도 이미징을 제어 </td></tr><tr><td> 열 교환기와 Cytomat 6001 </td><td> 써모 과학 </td><td> 51022222 </td><td> spImager에 부착된 </td></tr><tr><td> Ecoline RE207 </td><td> Lauda </td><td> RE207 </td><td> Cytomat에 부착된 </td></tr><tr><td> 회전 목마가있는 spImager </td><td> S &amp; P 로보틱스 INC </td><td> 사용자 정의 </td><td> 자동 고해상도 이미징 </td></tr><tr><td> FP12pins위한 로봇 핀 공구 고정구 </td><td> V &amp; P 과학 </td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96 X FP12 핀 </td><td> V &amp; P 과학 </td><td> FP12 </td><td> 50.4 mm 길이 17mm는 핀 길이를 노출 </td></tr><tr><td> 핀 도구에 대한 역 도킹 </td><td> V &amp; P 과학 </td><td> VP425 </td><td> 도킹 스테이션베이스 핀이 팬 건조를 허용하도록 제거 </td></tr><tr><td>브러시 청소 핀 </td><td> V &amp; P 과학 </td><td> VP425 </td><td> N / A </td></tr><tr><td> 복제 광택히 </td><td> 시그마 </td><td> R-2508 </td><td> 수동 핀 도구 </td></tr><tr><td> 뚜껑이있는 Nunc OmniTray </td><td> Nunc </td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96 잘 멸균 폴리스티렌 플레이트 </td><td> Greiner BioOne </td><td> 655,161 </td><td> N / A </td></tr><tr><td> 96 잘 멸균 폴리스티렌 뚜껑 </td><td> Greiner BioOne </td><td> 656,171 </td><td> N / A </td></tr></tbody></table> 표 3. 특정 시약 및 장비.

a quantitative fitness analysis workflow

양적 피트니스 분석 (QFA)는 병렬 1,2,3,4 재배 미생물 문화 fitnesses을 비교하기위한 실험 및 전산 작업 흐름이다. QFA은 단일 문화 focussed 관찰에 적용하지만, 독립적인 문화 수천까지 조사 게놈 전체 유전자 상호 작용이나 약물 화면에 가장 유용합니다. 중앙 실험 방법은 incubated 정기적으로 촬영되는 고체 한천 플레이트를 향해 독립 묽은 액체 미생물 문화의 접종이다. 매번 포인트에서 사진이 양적 피트니스 조치 얻을 수 있도록, 성장 곡선을 만드는 데 사용되는 정량 셀 밀도 견적, 제작, 분석하고 있습니다. 문화 fitnesses는 계량 및 유전적 상호 작용 강점이나 약물 감성 순위를 비교할 수 있습니다. 모든 치료의 문화 적합성에 대한 효과는 기판 한천 (예 : 작은 분자, 항생제 또는 영양분)에 추가하거나 접시 외부에 적용단조 (예 : 자외선 조사, 온도)이 QFA에 의해 계량하실 수 있습니다. QFA 흐름은 성장률이 96도 또는 200도 플레이트 리더에 평행 액체 문화의 분광 측정으로 얻은 것과 유사한 추정 생산하고 있습니다. 중요한 것은, QFA 같은 방법에 비해 훨씬 높은 처리량을 가지고 있습니다. QFA 배양 고체 한천 표면에 성장하므로 잘 교반이나 흔들림없이도 성장하는 동안 aerated됩니다. QFA 처리량 일부 합성 유전자 배열 (SGA) 전형 방법 5,6의만큼 높지 않을 것입니다. QFA 문화가 많이 한천로 주사되기 전에 희석되기 때문에 그러나, QFA 3 지수와 채도 단계를 포함하여보다 완벽한 성장 곡선을 캡처할 수 있습니다. 예를 들어, 성장 곡선 관찰 이전에 한 논의로 문화 더블링 시간이, 높은 정밀도와 직접 견적을 계산할 수 있습니다. 여기 소개특정 QFA 프로토콜 S. 수천에 적용 자동 접종, 배양 및 이미징 동안 로봇에 의해 처리됩니다 cerevisiae 문화. 이러한 자동화된 단계에는 처리량의 관련 감소와 함께 이에 상응하는, 수동 프로 시저로 대체하고, 우리는 또한 낮은 처리량 매뉴얼 프로토콜을 제시 할 수 있습니다. 동일한 QFA 소프트웨어 도구 중 워크플로우에서 촬영한 이미지에 적용할 수 있습니다. 우리는 광범위한 경험 신진 효모의 문화 QFA를 적용했습니다 S. 하지만 우리는 QFA는 분열 효모의 문화를 조사에 동일하게 유용 것으로 cerevisiae를 기대 S. pombe와 세균성 문화.

Watch this Scientific Journal Video about A Quantitative Fitness Analysis Workflow at JoVE.com

A Quantitative Fitness Analysis Workflow

양적 피트니스 분석 (QFA)는 미생물 문화 fitnesses을 계산하는 실험 및 전산 방법을 보완 시리즈입니다. QFA는 유전자 변이, 약물이나 미생물의 성장에있는 다른 적용 트리 트먼트의 효과를 추정하고있다. 단일 문화권의 focussed 분석에서 병렬 문화 수천 실험 스케일링을 설계할 수 있습니다.

양적 휘트니스 분석 워크플로우

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Research

JoVE Journal

Biology

14.3K 조회수.  Newcastle University Medical School. 양적 피트니스 분석 (QFA)는 미생물 문화 fitnesses을 계산하는 실험 및 전산 방법을 보완 시리즈입니다. QFA는 유전자 변이, 약물이나 미생물의 성장에있는 다른 적용 트리 트먼트의 효과를 추정하고있다. 단일 문화권의 focussed 분석에서 병렬 문화 수천 실험 스케일링을 설계할 수 있습니다.

비디오: A Quantitative Fitness Analysis Workflow

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

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

분열 효모의 양적 라이브 세포 형광 - 현미경 분석

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

시간 경과 비디오 현미경을 이용한 세포의 무작위 마이 그 레이션의 정량 분석

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

고급 3D 형광 현미경을 사용하여 자식 작용의 정량 분석

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

그레이 스케일 분석을 사용하여 마우스의 탈모의 정량 분석​​을위한 새로운 기술

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

히스톤 추출에서 데이터 분석에 : 상향식 질량 분석 사용 히스톤 번역 후 변형 분석을위한 완벽한 워크 플로우

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...