우리는 프로토콜에 대한 귀중한 의견을 제공 포터 그룹의 구성원을 감사드립니다. ;이 작품은 신경과 재단의 미국 아카데미에서 CMH하는 임상 연구 교육 교제, 일반 의료 과학 국립 연구소 (NIGMS의 친교에 의해 투자되었다 <a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a> JDR하기 (GM08169) ), NIH 국립 신경 장애 연구소에서 부여 뇌졸중 (NS054809) JDR에 루스 L. Kirschstein 내셔널 리서치 서비스 수상 (NS060392), NSF EFRI 부여 (0836017), 쿨터 재단 Translational 연구 상,와 JDR에 translational 연구 활동 (NS007480).

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CMH는 공개 아무것도 없습니다.<br /&gt; JDR와 SMP는 Axion Biosystems에 대한 컨설턴트를 지급됩니다.<br /이 문서와 동영상&gt; 제출 요금 및 비디오 처리 수수료는 우아하게 멀티 채널 시스템에 의해 후원되었다.

이 프로토콜은 방법을 문화에 대한 지침을 보여 주며의 연결을 미세 전극 배열에 네트워크뿐만 아니라에서 녹화 및 네트워크의 연결을 자극에 대한 소개를 유지합니다. MEAs에서 녹화 더 일반적인 문제 중 일부는 또한 MEA 문제 해결 섹션에서 논의되었습니다. 전반에 걸쳐 논의하고 자주 이러한 변화는 특정 실험 설계에서 요구하는 특정 조건을 최적화하기 위해 활용하는 범위 내에서 프로토콜 가끔 차이가 있습니다. 여기에 제시된 기록하고 자극 시스템 MCS의 preamp와 NeuroRighter 15 설계된 우리의 사용자 정의 구성이지만,의 연결 네트워크 인터페이스를 제공할 수있는 여러 다른 시스템이있다. 특정 설정을 선택하면 특정 실험 요구 사항에 따라 달라집니다. 간단한 녹음 및 자극의 예제 MEAs에 그러나 이러한 문화는 시냅스의 연결 활동과 네트워크 연결에 관한 복잡한 질문에 대한 통찰력을 제공하는 데 사용할 수있는이 프로토콜에서 제공되었습니다. 위에서 언급한 바와 같이 지속적인 작업이 세포 소성과 같은 프로세스를 연구하기 위해이 기술을 활용이며, 개발, excitotoxicity 및 neurodegeneration. 6-10 예를 들어, 우리의 실험실에서 최근 간행물 재현성 목표가 실시간 폐루프 자극과 함께 체외로 배우는 감독했다 현재의 연결을 네트워크 활동 3 응답. MEA의 문화가 자주 몹시 신경을 쓰는하고 섬세한 있지만, 그들의 단순 성과 접근성 (그대로 동​​물에 비해) 덕분에, 그들은 네트워크 수준의 연결 활동을 공부위한 강력한 모델을 제공합니다.

용품 <ul><li> 일회용 용기 상단 필터 (150ml, 0.2μm 기공 크기, Nalgene # 565-0020) </li><li> 배아 일 18 시간 초과 - 임신 여성의 쥐 </li><li> Microelectrode 배열 (B1, 프로토콜의 설명 참조) </li><li> Microelectrode 어레이 뚜껑 (프로토콜, B3의 설명 참조) </li><li> 나일론 셀 스트레이너 (40μm, BD 팔콘, # 352340) </li><li> 폴리 프로필렌 원뿔 튜브 (살균, 15ml) </li><li> 폴리스티렌 페트리 요리 (살균 35mm 및 100x15mm) </li><li> Pipet 팁 (살균, 20μl, 200μl와 1000μl) </li><li> Serological pipets (5ml) </li></ul> 장비 : <ul><li> 생물 안전 캐비닛 / 층류 흐름 후드 </li><li> 셀 카운터 </li><li> 원심 분리기 </li><li> 얼음 컨테이너 </li><li> 해부 가위와 집게 (소독) </li><li> 층류 후드에 현미경을 해부 </li><li> 휴대용 전기 pipetman </li><li> Hemacytometer </li><li> Isofluorane 및 가스 챔버 </li><li> 라이트 현미경 </li><li> 휴대용 pipetman (P - 20 (20μl), P - 200 (200μl), P - 1000 (1000μl)) </li><li> MEA 기록 시스템 (프로토콜, D1의 설명 참조) </li><li> 쥐 기요틴 </li><li> 트라이 - 가스 배양기 (프로토콜, B15에 대한 설명을 참조) </li><li> 후드에 부착된 흡인기로 진공 플라스크 </li><li> 와동 </li><li> 워터 목욕 (35-37 ° C) </li></ul> 솔루션 및 시약 : BSA 솔루션 : 확인 5% BSA 인산염의 (시그마 A - 9418)은 생리 산도 7.4와 0.2μm의 필터를 버퍼. 이 솔루션은 4 ° C 몇 달 동안 미리 준비하고 저장할 수 있습니다. 셀 매체 : 90 ML Dulbecco의 수정 이글 배지 (어바인 과학 9,024) 10 ML 말 혈청 (Gibco 16,050) 1ml 나트륨 pyruvate (Gibco 11360, 100mM), 250 μl GlutaMAX (Gibco 35,050) 및 인슐린 (최종 농도를 결합 2.5 μg / ML, 시그마 I - 5500)는 다음 0.2 μm의 filter.22 따뜻한 통과하고 사용하기 전에 인큐베이터에있는 세포 매체를 평형. papain 솔루션을 준비 해부 솔루션 : 23 <table border="1"><tr><td> 해부 솔루션 (100ml 주식) </td><td> 4, 순서 필터 0.22 μm의 및 상점을 결합 ° C. </td><td></td></tr><tr><td> 구성 요소 </td><td> 볼륨 ML </td><td> 최종 농도 (㎜) </td></tr><tr><td> 두 번 증류수 </td><td> 91.175 </td><td></td></tr><tr><td> 1 M MgCl 2 </td><td> 0.58 </td><td> 5.8 MM </td></tr><tr><td> 1 M CaCl 2 </td><td> 0.025 </td><td> 0.25 MM </td></tr><tr><td> 0.5 M의 HEPES </td><td> 0.32 </td><td> 1.6 MM </td></tr><tr><td> 0.5 % 페놀 레드 </td><td> 0.2 </td><td> (0.001 %) </td></tr><tr><td> 0.1 N NaOH </td><td> 0.2 </td><td> 0.2 MM </td></tr><tr><td> 2 M Na2SO4 </td><td> 4.5 </td><td> 90 MM </td></tr><tr><td> 1 M K2SO4 </td><td> 3.0 </td><td> 30 MM </td></tr><tr><td> 0.1 M Kynurenic 수 소산 </td><td> 1.0 </td><td> 1 ㎜ </td></tr><tr><td> 5 MM APV </td><td> 1.0 </td><td> 0.05 MM </td></tr></table> 이 솔루션은 4 ° C 몇 달 동안 미리 준비하고 저장할 수 있습니다. DNAse I : Invitrogen (# 18047019). 50μl aliquots는 -80 ° C.에 냉동 플래시 저장됩니다 나누어지는는 세포 분리 과정을 시작하기 전에 층류 후드에서 해동합니다. 행크의 균형 소금 솔루션 : 시그마 (# 4891). 1X 솔루션은 무균 0.2μm의 필터와 4에 저장된 ° C.는, 탈이온수에 준비가되어 있습니다 최대 절전 모드 솔루션 : culturing의 날에 준비합니다. 2 ML B - 27 보충제 (50X, Invitrogen # 17504)와 0.2 μm의에서 필터 98 ML L - 15 중간 (Invitrogen 11,415)을 결합합니다. Laminin 솔루션 : culturing의 날에 준비합니다. 0.02mg/ml의 최종 농도에 대한 980 μl 셀 매체 laminin의 20μl를 (1mg/ml, 시그마 L - 2020) 희석. laminin (1mg/ml) 20 μl aliquots는 -80 ° C에 보관하고 사용 직전 해동됩니다. 해동하면 벤치 위에 발생할 아니라 물 목욕과 같이 급속한 때로는 laminin의 겔화으로 이어질 수 있습니다 해동. Papain 솔루션 : 23 30-32로 해부 솔루션의 따뜻한 10 ML (위) ° C. 200 개가에게 papain을 추가 (시그마 P - 4762) 및 1.6 MG L - 시스테인 (시그마 C - 7880). 0.1 N NaOH의 약 14 μl와 7.4으로 산도를 조정합니다. 솔루션 CL까지 약 30 분 동안 품어귀, 필터 0.2 μm의, -80 15 ML의 폴리 프로필렌 살균 원뿔 튜브 및 저장 2 ML aliquots의 액체 질소로 동결 플래시 ° C. ° C 이전의 프로토콜을 시작하는 35-37에서 나누어지는를 녹여. Polyethyleneimine (PEI) 솔루션 : 13 PEI 100 μl (50 % W / V, 시그마 P - 3143)는 0.05 %의 최종 농도 W / V. 100 ML 나트륨 borate 버퍼 (0.1 M 산도 8.4, 시그마 B - 9876)에 희석입니다 0.2 μm의에서 멸균 필터. 이것은 사전에 준비하고 몇 달 동안 4 ° C에 저장할 수 있습니다. 무균 드 이온화된 물 : 압력솥 사용하기 전에 실온에 시원한 수 있습니다. 트립신 (EDTA (에틸렌 다이아 민 테트라 초산), Gibco 25,200로 0.25 %) 1ml aliquots는 -80 ° C.에 냉동 플래시 저장됩니다 Aliquots는 사용하기 전에 35-37 ° C의 물을 욕조에서 해동합니다.

how to culture, record and stimulate neuronal networks on micro-electrode arrays (meas)

지난 세기, 전세계 많은 neuroscientists이의 연결을 네트워크가 작동 방법을 이해하기 위해 자신의 인생을 헌신하고 왜 그들은 각종 질병에서 일하고 중지합니다. 연구 neuropathological 관찰, 유전자 라벨과 형광 현미경, 그리고 dissociated 뉴런과 슬라이스 준비 모두에서 세포 녹음을 포함합니다. 이 프로토콜은 마이크로 전극 배열 (또한 다중 전극 배열, MEAs)에 dissociated의 연결 문화를 성장과 관련된 또 다른 기술을 설명합니다. 다른 기술을 통해이 시스템을 사용하는 여러 장점이 있습니다. MEAs에 Dissociated의 연결을 문화 네트워크 활동이 배열 다중 전극을 통해 전기 자극을 시퀀스로 조절할 수있는 단순화된 모델을 제공합니다. 네트워크가 작은이기 때문에, 자극의 영향은 그대로 준비에 그런 경우가 아니라 관찰 지역으로 제한됩니다. 세포는 다양한 영상 기술을 모니터링하는 형태로 쉽게 변화를 만드는 monolayer 성장. 마지막으로, MEAs의 문화는 다른 culturing 기술과 본질적으로 어떤 명확한 시간 한계를 제거 체외에서 일년 이상 살아남을 수 있습니다. 1 전세계 우리의 실험실과 다른 사람의 연결 네트워크에 대한 중요한 질문을이 기술을 활용하고 있습니다. 이 프로토콜의 목적은, 돌보는, 설정에서 녹음 및 전기 MEAs의 문화를 자극하기 위해 필요한 정보를 제공하는 것입니다. 네트워크에서 생리학 관련 질문을과 더 나은 이해를 선도하는 세포 수준의 수단을 제공 체외 네트워크에서 두뇌 기능과 장애.

Watch this Scientific Journal Video about How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs at JoVE.com

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

이 프로토콜은 설정을 돌보는에서 녹음 및 전기 MEAs의 문화를 자극하기 위해 필요한 정보를 제공합니다.<em&gt; 체외에서</em&gt; 네트워크 네트워크 및 두뇌 기능과 기능 장애의 더 나은 이해를 선도하는 세포 수준에서 생리학 관련 질문을위한 수단을 제공합니다.

어떻게 문화, 기록 및 미세 전극 배열에의 연결을 네트워크를 자극 (MEAs)에

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

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Biology

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

54.3K 조회수.  Emory University School of Medicine. 이 프로토콜은 설정을 돌보는에서 녹음 및 전기 MEAs의 문화를 자극하기 위해 필요한 정보를 제공합니다. 체외에서 네트워크 네트워크 및 두뇌 기능과 기능 장애의 더 나은 이해를 선도하는 세포 수준에서 생리학 관련 질문을위한 수단을 제공합니다.

비디오: How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

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 ...

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Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

병아리 예 비켜 문화와 예 비켜 CAM 어세이 : 그게 정말로 작동하는 방법

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

에 박테리아 기능 네트워크 및 경로를 매핑<em&gt; 대장균</em&gt; 합성 유전자 배열을 사용하여

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

다중 Chemostat 배열의 설계 및 사용

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

기록 개인과 성인 Zebrafish의 떼에 자동 3D 추적 시스템을 사용하여

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

기술은 유도 및 정량화 세포 노화하기

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

아디포세포 기능에 대한 단백질 및 마이크로 RNA 변조의 영향을 연구하는 세포 배양 모델

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

가중 상관 관계 네트워크에 따라 다른 서식지에서 루트 미생물의 발산

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...