이 논문에 설명된 연구는 캐나다 혁신, 자연 과학 및 공학 연구 위원회, 서스 캐처 원 대학, 서스 캐처 원 정부, 서부 경제 다각화 캐나다, 캐나다 국립 연구위원회 및 캐나다 보건 연구소의 캐나다 혁신 재단이 지원하는 캐나다 광원의 BMIT 시설에서 수행되었습니다. 저자는 캐나다 광원의 빔 라인 과학자, 특히 아담 웹, 데니스 밀러, 세르게이 가질로프, 닝 주 스카이스캔 SRμCT 및 백색 빔 현미경 시스템의 설정 및 문제 해결에 도움을 주셔서 감사합니다. 우리는 또한 이 연구 결과에 대한 cadaveric 견본에 접근을 위한 Toledo 의과 대학의 대학 및 박사 제프리 웬스트루프에서 베스 달젤에게 감사드립니다. JM Andronowski는 애크런 대학과 형사 사법 목적 부여를위한 법의학 과학의 국립 사법 연구 개발 연구소 (2018-DU-BX-0188)에서 제공하는 창업 연구 기금을 통해 지원됩니다. RA 데이비스는 애크런 대학에서 제공하는 대학원 보조에 의해 지원됩니다. 코링 과 톱질에 사용되는 장비와 공급은 아크론 대학과 NSF가 CW 홀리오크에 EAR-1624242를 부여하는 스타트업 기금에 의해 구입되었습니다.

<ol>
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저자는 공개 할 것이 없습니다.

제한된 FOV 설정을 갖춘 고해상도 SRμCT 이미징을 위한 균일한 및 원통형 피질 뼈 코어 샘플을 조달하기 위한 포괄적이고 표준화된 프로토콜은 없었습니다. 여기에 자세히 설명된 프로토콜은 SRμCT 이미징에 대한 일관되게 크기의 피질 뼈 코어 샘플을 조달하는 방법과 미세 건축 데이터의 후속 정확한 시각화 및 추출에 대한 포괄적인 자습서를 제공함으로써 무효화를 채웁니다. 당사의 프로토콜은 ?...

이미징 기술의 최근 발전으로, 지금 매우 높은 해상도의 X 선 이미징 데이터를 취득하는 것이 가능합니다. 데스크탑 마이크로 CT(μCT) 시스템은 비파괴적 특성1로인해 방수 골격을 이미징하는 현재 표준입니다. 그러나 피질 뼈의 미세 구조 적 특징을 이미징 할 때 μCT 사용은 더 제한적이었습니다. 해상도 제약으로 인해 데스크톱 시스템은 골세포 라쿠나에 같은 피질 모공보다 작은 미세 구조 적 특징을 이미지하는 데 필요한 해상도를 달성할 수 없습니다. 이 응용 프로그램의 경우 SRμCT는 이러한 시스템1의해상도가 커지므로 이상적입니다. 예를 들어, 생물 의학 이미징 및 치료 (BMIT) 빔 라인2에 캐나다 광원 (CLS)에서 실험은 0.9 μm만큼 작은 복셀이미지를 생산하고있다. 이전 연구1,3,4,5는 인간 긴 뼈(그림 1)에서피질 뼈 표본으로부터 프로젝션및 후속 3차원 (3D)을 획득하기 위해 이 해상도를 사용하여 골구세포 라부나 밀도4,6,7,9및라쿠나 모양및 크기3의 변화및 성간 사이의 변화. 추가 연구는 인간에서 골연 밴딩의 존재를입증했다 10,이전에 법의학 인류학 문헌에서 비 인간 포유동물과 연관된 것으로 인식 되는 현상.
뛰어난 해상도를 달성하기 위해 X선 빔은 최대 시편 크기를 직경 이내로 제한하는 시야(FOV) 내에 미세하게 초점을 맞추어야 합니다. 현재, 이러한 제한을 충족하는 뼈 샘플 조달을 요약하는 문헌에 설명된 포괄적이고 표준화된 절차는 없었습니다. FOV 내의 중심 표본은 1) 이미징 중에 180°를 회전할 때 샘플이 중심을 유지하고 2) 스캔 아티팩트가 이미지 잘림이 없기 때문에 제한되도록 하는 것이 중요합니다. 즉, FOV 외부의 시료의 어떤 부분도 FOV 내부의 초점으로 들어가는 빔을 방해하지 않는다. 이 경우 재구성 알고리즘은 완전히 올바른 재구성에 필요한 일부 감쇠 데이터가 박탈됩니다. 360° (전체 회전) 스캔빔 경화의 영향을 최소화하지만 이미징 중 정렬 불량 및 샘플 움직임으로 인한 아티팩트를 증가시키는 것은 주목할 가치가 있습니다. 따라서 360° 검사는 일반적으로 더 깨끗한 데이터를 생성하지만 이미징 시간이 두 배가되므로 실험 비용과 데이터 품질 간의 절충안을 해결해야 합니다.
뼈 화상 진찰 실험의 중요하고 수시로 간과되는 양상은 스캐닝의 앞에 수행된 정확하고 복제가능한 견본 준비 기술입니다. SRμCT 방법을 실험에 통합하는 연구는 샘플링 프로토콜을 간략하게 언급하지만 저자는 표본을 수집하는 데 사용되는 특정 방법론에 대해 거의 또는 전혀 세부 사항을 제공하지 않습니다. 많은 연구는 임의 차원의 직선 뼈 블록을 절단 언급하지만, 일반적으로사용되는도구 또는 포함 재료에 대한 자세한 정보를 제공하지3,4,10,11,12,13,14. 일부 연구자들은 일반적으로 핸드헬드 로터리 도구(예를 들어, Dremel)를 사용하여 관심 영역(ROI)3,4,10,11, 12,13,14에서뼈의 직선 블록을 제거한다. 이 방법은 FOV보다 더 큰 균일하게 크기의 샘플을 생성하여 아티팩트 및 이미지 잘림 스캔가능성을 높입니다. 이러한 표본은 종종 정밀 다이아몬드 웨이퍼 톱 (예 : Buehler Isomet)을 사용하여 추가 정제가 필요합니다. 일관된 차원(200/mm)으로 샘플을 조달하는 것은 획득된 데이터 집합이 최고 품질이고 후속 결과가 복제가능한지 확인하는 데 중요합니다.
샘플 조달 방법론의 제한된 보고는 이전 연구에서 수행된 방법을 사용 및/또는 검증하려고 할 때 어려움을 더합니다. 현재 연구원은 샘플링 절차에 대한 자세한 내용은 저자에게 직접 문의해야 합니다. 여기에 자세히 설명된 프로토콜은 생물 의학 연구자들이 철저히 문서화되고 복제되고 비용 효율적인 샘플링 기술을 제공합니다. 이 문서의 주요 목적은 미세 건축 데이터의 정확한 시각화 및 추출을 위해 밀 드릴 프레스 및 다이아몬드 코링 비트를 사용하여 일관되게 크기의 피질 뼈 코어 샘플을 조달하는 방법에 대한 포괄적 인 자습서를 제공하는 것입니다. 이 방법은 고압 암석 역학15,16,17,18,19의단단한 재료 블록에서 균일하고 작은 직경 (1-5 mm) 실린더를 일상적으로 수집하는 데 사용되는 절차에서 수정됩니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1367px;" width="1367">
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			<td>Model: JMD-15, benchtop drill presses are suitable substites, but typically lack a translatable machine table for positioning samples beneath the drill stem</td>
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			<td height="21" style="height:21px;width:215px;">Fine-tipped forceps</td>
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			<td>75x50mm slides, 1mm thick</td>
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			<td>Large enough to hold 75x50mm glass slides</td>
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			<td height="42" style="height:42px;width:215px;">Hot plate capable of reaching 140 &#176;C</td>
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			<td height="21" style="height:21px;width:215px;">NRecon</td>
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			<td>Download and install at https://www.bruker.com/products/microtomography.html</td>
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</table>

a sectioning, coring, and image processing guide for high-throughput cortical bone sample procurement and analysis for synchrotron micro-ct

뼈는 인간의 수명 동안 구조에 변화 하는 동적 및 기계적으로 활성 조직. 뼈 리모델링 과정의 제품은 기존의 2차원 기술을 사용하여 실질적으로 연구되었습니다. 데스크톱 마이크로 컴퓨팅 단층 촬영(μCT) 및 싱크로트론 방사선 마이크로 컴퓨터 단층 촬영(SRμCT)을 통한 X선 이미징 기술의 최근 발전은 다른 3D 이미징 기술(예를 들어, SEM)보다 더 큰 시야(FOV)의 고해상도 3차원(3D) 스캔을 획득하여 인간 내 마이크로코픽 구조의 보다 완전한 그림을 제공한다. 그러나 표본은 FOV 내에서 정확하게 중심을 두어 데이터 분석에 영향을 미치는 것으로 알려진 줄무늬 아티팩트의 모양을 제한해야 합니다. 이전 연구는 고르지 못한 가장자리 또는 이미지 잘림으로 인해 이미징 아티팩트를 초래하는 불규칙한 모양의 직선 골 블록의 조달을 보고했습니다. 우리는 인간 페모라의 전방 양으로부터 SRμCT 실험을 위해 일관되게 크기의 피질 뼈 코어 표본을 조달하기 위해 지질 학적 샘플링 프로토콜 (coring)을 적용했습니다. 이 코링 방법은 효율적이고 조직에 최소한의 파괴적입니다. 균일한 원통형 샘플을 생성하여 회전 시 동포메트릭이 되고 스캔 전반에 걸쳐 X선 빔에 대한 균일한 경로 길이를 제공하는 특성상 이미징 아티팩트를 감소시합니다. 코어및 불규칙한 모양의 샘플의 X선 지형 데이터의 이미지 처리는 피질 뼈 미세 아키텍처의 시각화 및 분석을 개선하는 기술의 잠재력을 확인합니다. 이 프로토콜의 목표는 다양한 유형의 고해상도 뼈 이미징 실험에 적응할 수 있는 피질 뼈 코어추출을 위한 신뢰할 수 있고 반복가능한 방법을 제공하는 것입니다. 이 작업의 가장 중요한 목표는 저렴하고 일관되며 간단하며 SRμCT를 위한 표준화된 피질 뼈 조달을 만드는 것입니다. 이 절차는 생물학적 인류학, 지구과학 또는 물질 과학과 같은 단단한 복합 물질을 일반적으로 평가하는 관련 분야의 연구원에 의해 더 적응될 수 있습니다.

설명된 코어 샘플링 방법은 매우 효과적이고 효율적인 것으로 입증되었습니다. 이 프로토콜을 사용하여 코링 시편은 CLS BMIT-BM 빔라인2의실험을 위해 일관되게 크기의 샘플을 조달할 수 있었으며, FOV는 1.49 μm 복셀 크기로 ~2mm입니다. 코어 직경의 일관성을 검증하기 위해 인간 전방 대퇴코어(n=69)의 하위 집합의 길이(위,중간, 아래)를 따라 세 가지 측정을 수행하였다. 코어?...

Watch this Scientific Journal Video about A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT at JoVE.com

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

우리는 인간 페모라의 전방 측면에서 SRμCT 실험을 위해 균일한 크기의 피질 뼈 표본을 조달하기 위해 지질학적(coring) 샘플링 프로토콜을 사용했습니다. 이 방법은 최소한의 파괴적이고 효율적이며, 불규칙한 샘플 모양에서 이미징 아티팩트를 최소화하고 미세 건축 시각화 및 분석을 향상시키는 원통형 표본을 초래합니다.

싱크로트론 마이크로 CT를 위한 고처리량 피질 뼈 샘플 조달 및 분석을 위한 단면, 코링 및 이미지 처리 가이드

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

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JoVE Journal

Biology

5.0K 조회수.  The University of Akron. 우리는 인간 페모라의 전방 측면에서 SRμCT 실험을 위해 균일한 크기의 피질 뼈 표본을 조달하기 위해 지질학적(coring) 샘플링 프로토콜을 사용했습니다. 이 방법은 최소한의 파괴적이고 효율적이며, 불규칙한 샘플 모양에서 이미징 아티팩트를 최소화하고 미세 건축 시각화 및 분석을 향상시키는 원통형 표본을 초래합니다.

비디오: A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

연구

생물학

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

성인의 준비 Drosophila</em씬 Sectioning 및 현미경 분석을위한> 눈

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

비전문가를위한 계통 발생에 대한 실용 가이드

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

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 ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사​​용자 친화적 인 소프트웨어 응용 프로그램

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

대량 세포 계측법 분석을위한 샘플 준비

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

높은 처리량, 광물 조직의 멀티 이미지 Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

싱크로트론 마이크로 CT를 위한 고처리량 피질 뼈 샘플 조달 및 분석을 위한 단면, 코링 및 이미지 처리 가이드

요약

더 많은 비디오 탐색

이 비디오의 챕터

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

성인의 준비 Drosophila 눈

비전문가를위한 계통 발생에 대한 실용 가이드

펩티드 에피토프의 정량 분석을위한 높은 처리량 MHC II 결합 분석

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사용자 친화적 인 소프트웨어 응용 프로그램

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

대량 세포 계측법 분석을위한 샘플 준비

높은 처리량, 광물 조직의 멀티 이미지 Cryohistology

싱크로트론 마이크로 CT를 위한 고처리량 피질 뼈 샘플 조달 및 분석을 위한 단면, 코링 및 이미지 처리 가이드

요약

더 많은 비디오 탐색

이 비디오의 챕터

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

성인의 준비 Drosophila 눈

비전문가를위한 계통 발생에 대한 실용 가이드

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

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사​​용자 친화적 인 소프트웨어 응용 프로그램

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

대량 세포 계측법 분석을위한 샘플 준비

높은 처리량, 광물 조직의 멀티 이미지 Cryohistology

펩티드 에피토프의 정량 분석을위한 높은 처리량 MHC II 결합 분석

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사용자 친화적 인 소프트웨어 응용 프로그램