<html> 여기 루카스 [1-3]에 관련된 실험 절차를 논의합니다. 루카스의 개념의 증거를 보여주면 우리는 전체 혈액 샘플에 대한 이미징 프로세스를 설명합니다. A. 이미징 설정 루카스 이미징 플랫폼은 특히 자원 제한 설정에 대한 기존의 포인트 - 오브 - 케어 cytometry 의료 진단 도구에 대한 비용 효율적인 소형 대안을 제공하는 중요한 이점을 전시하고 있습니다. 오히려 세포의 이미지를 감지하는 것보다 루카스 대신 배경 조명 각 세포에서 흩어져있는 빛의 간섭에 의해 만들어진 세포의 디지털 홀로그램을 캡처합니다. 조명의 부분 공간 일관성에주의 제어 홀로그램 기록을 사용하는 것이 중요합니다. 1. 디지털 센서 어레이 루카스 플랫폼은 디지털 개인 휴대 홀로그램을 기록하는 광전자 센서 배열을 사용합니다. 사용할 수 있습니다 또는 보완 금속 - 산화물 - 반도체 칩 (MT9P031, 마이크론 CMOS, 샘플 모델),이를 위해 몇 가지 장치 (카이 - 11002, KAF - 39000, 코닥의 샘플 모델 CCD) 부과. 각각 활성 10cm2의 FOV, 18cm2, 그리고 24.4 mm2와 코닥을위한 픽셀 크기는 두 장치, 카이 - 11002, KAF - 39000을 충전하고, 마이크론 CMOS 이미지 센서는 9 μm의, 6.8 μm의 및 2.2 μm의 수 있습니다. [1-2]. 2. 라이트 소스 대부분의 다른 미세한 이미징 modalities과는 달리, 루카스는 따라서 레이저, 심지어 간단한 발광 다이오드 (LED) 조명에 사용할 수있는 필요하지 않습니다. 조정할 수있는 파장의 조명을 사용하기 위해, 우리는 섬유의 다발 (77564, 뉴 포트)과 핀홀 구성된 표준 등급 융합 실리카 섬유와 함께 제논 램프 (코너 T260, 뉴 포트 주식 회사)로 단색을 활용할 수 있습니다 ~ 100 μm의 직경은 센서 표면 위 ~ 50-10센티미터에 위치하고 있습니다. 이 조정할 수있는 파장 조명 구성 세포의 홀로그램 서명 조정할 수있는 유연한 플랫폼을 제공하고, 하이브리드 디지털 서명은 더 나은 특성화 정확성과 특이성에 대한 잡음 비율로 신호를 향상시키기 위해 합성 수 있습니다. [3] B. 샘플 준비 및 이미징 영상 - 칩을 기반으로 루카스의 개념 증명은 아래 설명된대로 이기종 솔루션을 사용하여 시연합니다. 유사한 프로토콜은 다양한 세포 유형 [1-3]에 적용할 수 있습니다. 1. 전체 혈액 희석과 이기종 솔루션의 준비 <ol><li> 프로세스를 시작하려면, 전체 혈액 샘플과 다양한 직경 (5, 10, 20 μm의. 듀크 과학)의 폴리스티렌 microbeads를 얻을하고, 실온에 모두 가져. </li><li> 멸균 5 ML의 폴리 프로필렌 튜브에 희석제로서 RPMI 1640 고전 액체 미디어 (피셔 과학) 2 ML을 추가합니다. </li><li> 30 분 전체 혈액 샘플을 잠복기 후, sedimented 적혈구 시료 10 μL를 피펫 및 RPMI 솔루션으로 그것을 전송합니다. </li><li> 적혈구 RPMI 희석에 20 μL 폴리스티렌 microbeads의 총 볼륨을 추가하고 부드럽게 pipetting 또는 저어 막대와 자기 활동가를 사용하여 셀 솔루션을 선동하다. </li><li> 포셉를 사용하여 솔루션을 통해 두 번째 동일한 커버 슬립을 풀 장소 표지에 5-15 μL 이기종 솔루션의 총 볼륨을 넣으십시오. 예제는 균일 두 커버 전표 사이에 끼워 넣으면됩니다. </li><li> 그런 다음, 진공 펜 (NT57 - 636, 에드먼드 광학)를 사용하면 이미지에 대한 센서 배열의 활성 영역에 샘플을로드합니다. </li></ol> 2. 전체 혈액 스테 이닝 <ol><li> 0.1 %의 혼합물의 준비를 시작으로 전력이 유형의 순수 Eosin Y (MW = 691.85, Acros의 생명체), 아연없는 순수한 새로운 메틸렌 블루 염색 (MW = 347.90, Acros의 유기물을)를 사용하여 Eosin Y와 희석 새로운 메틸렌 솔루션 버퍼 그리고 칼륨 Oxalate 일수화물 (99.0 %의 시약 ACS, Acros의 생명체)가 있으며, 각 솔루션은 0.45 μm의 기공 크기 Syringless 필터 (왓먼) 수성 백혈구의 얼룩을 위해 함께 정화됩니다. </li><li> 그런 다음, 부드럽게 2 분 동안 자기 활동가를 사용하여 폴리 프로필렌의 비커에 1시 1분의 음량 비율에서 전체 혈액 표본과 함께 해산 수성 얼룩 시약을 흔들어 10 분 알을 품다. </li><li> 5 ML의 폴리 프로필렌 튜브에 50 μL의 스테인드 혈액 솔루션을 전송하고 1:200의 볼륨 비율을 확보하기 위해 1.95 ML의 RPMI 1640 고전 액체 용액 (피셔 과학)로 희석. 그런 다음, 와동 30 초 희석. 다른 희석 비율도이 단계에서 도입하실 수 있습니다. </li><li> 커버 전표 사이 또는 microfluidic 저수지 내에서 10-100 μL의 스테인드 세포 솔루션을 피펫. 그런 다음, 진공 펜 (NT57 - 636, 에드먼드 광학)를 사용하여, 센서 배열의 활성 영역에 표본을 넣으십시오. </li></ol> 대표 결과 ve_content &quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="그림 1" border="0" /> 그림 1 : 루카스가 정확하게 계산하고 렌즈, 레이저 또는 기타 대형 광학 구성 요소를 사용하지 않고 2D 홀로그램 서명을 기반으로 다양한 세포 및 미세 물체의 디지털 차별화 수 있습니다. 다양한 마이크로 개체의 특성 루카스 서명이 그림 그림하고 40X 객관적인 렌즈로 얻은 기존의 현미경 이미지를 비교하고 있습니다. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="그림 2" /> 그림 2 : 적혈구, 10um, 5um, 3 음 입자를 포함하는 이기종 혼합 (왼쪽) RAW 이미지. 보기의 동일한 필드 (오른쪽) 완전 자동 루카스 특성화 결과는 그림입니다. 결정 알고리즘은 3um 비즈 같은 노이즈 비율 고밀도 지역뿐만 아니라 낮은 신호 입자를 특성화로 강력한입니다. <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="그림 3" border="0" /> 그림 3 : 루카스 사용자 인터페이스가 그림입니다. 자바 기반의 루카스 소프트웨어는 센서 픽셀 크기 또는 빛의 파장 등 다양한 실험 조건에 대한 입력을 허용합니다. 이미지를보기의 특정 분야의 선택도 만들 수 있으며, 대상 세포 패턴은 통계 셀 그림자 라이브러리를 빌드하기 위해 사용자가 정의할 수 있습니다. 인수 루카스 이미지는 다음이 훈련 데이터 (즉, 세포 그림자 라이브러리)와 표시 (계산) 이미지가 사용자에게 표시됩니다에 따라 특징 수 있습니다. 카운트 결과의 통계도 추가 분석을위한 XML 파일로 저장됩니다.

<ol>
	<li>Seo, S., Su, T., Tseng, D. K., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Lensfree+Holographic+Imaging+for+On-Chip+Cytometry+and+Diagnostics".">"Lensfree Holographic Imaging for On-Chip Cytometry and Diagnostics".</a> Lab Chip. , (2009).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="High-throughput+Imaging+and+Characterization+of+a+Heterogeneous+Cell+Solution+on+a+Chip"+.">"High-throughput Imaging and Characterization of a Heterogeneous Cell Solution on a Chip" .</a> Biotechnology and Bioengineering. , (2008).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Multi-color+LUCAS:+Lensfree+on-chip+cytometry+using+tunable+monochromatic+illumination+and+digital+noise+reduction".">"Multi-color LUCAS: Lensfree on-chip cytometry using tunable monochromatic illumination and digital noise reduction".</a> Cellular and Molecular Bioengineering. , (2008).</li></ol>

우리는 루카스 플랫폼이 정확하게 계산하고 홀로그램 서명을 기반으로 한 칩에 다양한 micro-objects/cells을 식별할 수있는 그림, 그리고 포인트의 케어 의료 진단 및 높은 처리량 세포 생물학을위한 유망한 도구를 제공합니다. 정확하게 기록된 홀로그램 패턴을 처리하기 위해, 우리는 사용자 지정 개발 루카스 결정 소프트웨어를 구현했습니다. 루카스 이미지의 훈련에 의해 만들어진 통계 회절 이미...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAI-11002</td>
<td>&nbsp;</td>
<tr>
<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAF-39000</td>
<td>&nbsp;</td>
<tr>
<td>Complementary Metal-Oxide-Semiconductor (CMOS)</td>
<td>&nbsp;</td>
<td>Micron</td>
<td>MT9P031</td>
<td>&nbsp;</td>
<tr>
<td>Xenon Lamp</td>
<td>&nbsp;</td>
<td>Newport Corp.</td>
<td>Cornerstone T260</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pen</td>
<td>&nbsp;</td>
<td>Edmund Optics</td>
<td>NT57-636</td>
<td>&nbsp;</td>
<tr>
<td>5, 10, and 20 &mu;m Microbeads</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>4000 Series</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1640</td>
<td>&nbsp;</td>
<tr>
<td>Pure Eosin Y</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=691.85</td>
<td>&nbsp;</td>
<tr>
<td>New Methylene Blue(NMB) Dye</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=347.90</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Oxalate Monohydrate</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>99.0% Reagent ACS</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics

다른 렌즈와 광학 구성 요소가 함께 작동 객관적인 렌즈의 사용에 의해 종래의 광학 현미경 이미지 세포. 꽤 효과가 있지만,이 고전적인 방법은 microfluidics에있는 예술의 고급 상태와 호환되도록 이미징 플랫폼의 소형화에 대한 특정 제한 사항이 있습니다. 이 보고서에서는, 우리는 루카스를 칭했다 lensless 온 칩 이미지 개념 실험 세부 사항 (을 소개합니다<strong<em&gt; L</em</strong&gt; ensless<strong<em&gt; U</em</strong&gt; ltra 전체 뷰 필드<strong<em&gt; C</em</strong&gt; 엘 모니터링<strong<em&gt;</em</strong에 따라&gt; rray 플랫폼<strong<em&gt; S</em</strong만큼 큰 스팬 수있는보기의 초고 다양한 분야를 통해 이미지 이종 세포 솔루션을 어떤 현미경의 목표 또는 다른 대형 광학 부품을 필요로하지 않습니다&gt; 헤도우 영상) ~ 18cm<sup&gt; 2</sup&gt;. 또한, 기존의 현미경과는 달리, 루카스 이미지가 ~ 9 ML 샘플 볼륨에 해당 환기 시켰을 뿐일세 필요없이 ~ 5mm의 깊이 - 오브 - 분야 이상의 관심의 이종 세포 솔루션하실 수 있습니다. 이 영상 플랫폼 유형, 솔루션 내에 개수와 세포의 상대적 위치를 확인할 수 있습니다 각보기의 해당 분야 내에서 관심있는 세포 및 이러한 세포 그늘의 자동 디지털 처리의 그림자 (즉, lensless 디지털 홀로그램) 기록합니다. 그것이 어떤 대형 광학 부품이나 기계 검사 단계를 필요로하지 않기 때문에 그것은 동시에 치료 진단 도구 특히 지점에 대한 매우 중요 비용을 줄일 수있는 상당히 소형 플랫폼을 제공합니다. 또한,이 플랫폼의 영상 처리량이 높은 처리량 세포 생물학 실험을위한 매우 중요한 것일 수 종래의 광학 현미경보다 더 크기의 명령입니다.

Watch this Scientific Journal Video about Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics at JoVE.com

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

세포의 Lensfree 온 - 칩 이미징 및 특성은 그림이다. 이 온칩 세포 이미징 접근 방식은 자원 가난한 설정을 위해 특히 적합, 의료 진단 및 높은 처리량 세포 생물학 애플 리케이션을위한 소형 및 비용 효율적인 도구를 제공합니다.

셀 Lensless 온 - 칩 영상은 높은 처리량 세포 생물학 및 의료 진단을위한 새로운 도구를 제공합니다

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

lensless-on-chip-imaging-cells-provides-new-tool-for-high-throughput

Research

JoVE Journal

Biology

11.9K 조회수.  University of California, Los Angeles. 세포의 Lensfree 온 - 칩 이미징 및 특성은 그림이다. 이 온칩 세포 이미징 접근 방식은 자원 가난한 설정을 위해 특히 적합, 의료 진단 및 높은 처리량 세포 생물학 애플 리케이션을위한 소형 및 비용 효율적인 도구를 제공합니다.

비디오: Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

기관 절개술 : 폐에 줄기 세포의 이식을위한 방법

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

연구

생물학

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

세포 생물학을위한 그라디언트 생성 Microfluidic 장치

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

에서 지방 질량의 생화학 및 높은 처리량 미세 평가<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

반핵 항체 HEP-2 세포를 사용하여 상영

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

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 ...

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 ...

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a systems-level. Genome-scale metabolic models may include gaps as well as reactions that are unverified experimentally. Reconstructed models of newly isolated microalgal species will result in weaknesses due to these gaps, as there is usually sparse biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology is an effective, high-throughput method that functionally determines cellular metabolic activities&#160;in response to a wide array of entry metabolites. Combining the high throughput phenotypic assays with metabolic modeling can allow existing metabolic network models to be rapidly reconstructed or optimized by providing biochemical evidence to support and expand genomic evidence. This work will show the use of PM assays for the study of microalgae by using the green microalgal model species Chlamydomonas reinhardtii as an example. Experimental evidence for over 254 reactions obtained by PM was used in this study to expand and refine a genome-scale C. reinhardtii metabolic network model, iRC1080, by approximately 25 percent. The protocol created here can be used as a basis for functionally profiling the metabolism of other microalgae, including known microalgae mutants and new isolates.

This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model.

미세 조류의 모델 개선을 위한 높은 처리량 신진 대사 프로파일링

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

성장과 유전자 발현의 이질성을 측정하는 마이크로콜로니의 높은 처리량 라이브 이미징

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

세포 DNA 손상 평가를 위한 고처리량 혜성 분석 접근법

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

오가노이드의 배양 및 3D 이미징을 위한 고처리량 플랫폼

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...