이 작품은 웨스트 버지니아 대학의 메리 Babb 랜돌프 암 센터에서 기금 기금에 의해 지원되었다. 우리는 초기 조언과 도움을 Susette 뮐러 (조지 타운 대학교 (Georgetown University))와 로라 켈리 감사드립니다. 웨스트 버지니아 대학 현미경 영상 시설의 사용은 (메리 Babb 랜돌프 암 센터 및 NIH 보조금 P20 RR16440, P30 RR032138와 P30 GM103488에서 지원) 굉장히 인정하고 있습니다.

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관심 없음 충돌이 선언 없습니다.

세포 외 기질 굴욕 세포를 시각화 할 수있는 능력은 세포 침략의 초기 단계에서 사용 된 분자 메커니즘을 발견에서 도왔습니다. 1980 년대 초 4,14,15에 웬 - 티엔 첸에 의해 개척 이후 미세 분석을위한 유리 coverslips에 코팅 휘황 표시 세포 단백질은 세포 유형의 다양한 범위에 걸쳐 invadopodia 기능을 평가의 기본 기술로 떠오르고있다. 소정의 프로토콜이 일에 비슷한 기존의 형광 및 공 촛점 현...

<table border="1"><tbody><tr><td> 시약 / 악기의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 젤라틴 </td><td> 시그마 </td><td> G1890 </td><td> 돼지 피부 </td></tr><tr><td> 자당 </td><td> 어부 </td><td> BP220 </td><td></td></tr><tr><td> 12mm coverslips </td><td> 어부 </td><td> 50-121-5159 </td><td></td></tr><tr><td> 24 잘 접시 </td><td> 어부 </td><td> 08-772-1 </td><td> BD 팔콘 </td></tr><tr><td> 질산 </td><td> Ricca </td><td> R5326000 </td><td> 20 % 솔루션 </td></tr><tr><td> 폴리-L-라이신 </td><td> 전자 현미경 과학 </td><td> 19320-B </td><td> 0.1 % 용액 </td></tr><tr><td> 글 루타 알데히드 </td><td> 시그마 </td><td> G7526 </td><td> 8 % 솔루션 </td></tr><tr><td&gt; 오레곤 그린 488 - 복합 젤라틴 </td><td> Invitrogen </td><td> G-13186 </td><td></td></tr><tr><td> 나트륨 borohydride </td><td> 시그마 </td><td> 213462 </td><td></td></tr><tr><td> 트리톤 X-100 </td><td> 어부 </td><td> BP151 </td><td></td></tr><tr><td> rhodamine-phalloidin </td><td> Invitrogen </td><td> R415 </td><td></td></tr><tr><td> 반 cortactin (클론 4F11) </td><td> Millipore </td><td> 05-180 </td><td></td></tr><tr><td> 안티 - 플래그 항체 </td><td> Millipore </td><td> MAB3118 </td><td></td></tr><tr><td> 알렉사 형석 647 염소 안티 - 마우스 IgG </td><td> Invitrogen </td><td> A21235 </td><td></td></tr><tr><td> 골드 antifade을 연장 </td><td> Invitrogen </td><td> P36930 </td><td></td></tr><tr><td> LSM 510 공 촛점 현미경 </td><td> Zeiss </td><td></td><td></td></tr><tr><td> ImageJ 소프트웨어 </td><td> 공개 </td><td></td><td> <a href="http://www.macbiophotonics.ca/imagej/" target="_blank">http://www.macbiophotonics.ca/imagej/</a> </td></tr><tr><td> AutoQuant X2.2 소프트웨어 </td><td> 미디어 인공 이식 </td><td></td><td></td></tr><tr><td> NIS 요소 소프트웨어 </td><td> 니콘 </td><td></td><td></td></tr></tbody></table>

quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts

지역 조직에 세포 침공은 개발과 항상성에서 중요한 과정입니다. Malregulated 침공과 이후의 세포 운동은 염증, 심장 혈관 질환과 종양 세포 전이 한 등 여러 가지 병적 인 프로세스의 특징입니다. 상피 또는 내피 지하 막에서 세포 외 기질 (ECM)의 구성 요소 Focalized proteolytic 저하는 세포 침공을 시작으로 중요한 단계입니다. 종양 세포에서 체외 분석에 광범위한는 ECM 저하가 invadopodia 2,3를 칭했다 복부 고를 풍부한 막 밀어내는 구조에 의해 수행되는 결정을 내 렸습니다. 그들은 매트릭스 metalloproteinases (MMPs)의 행동을 통해 ECM 분석을 검토 ECM에 가까운 동격의 Invadopodia 양식. invadopodia을 형성하는 종양 세포의 능력은 바로 지역의 기질 및 관련 혈관 구성 요소를 3으로 침입 할 수있는 능력과 함께 연결합니다. _content는 &quot;유리 coverslips에 코팅 염료 표시 매트릭스 단백질을 사용하여 형광 현미경에 의한 세포의 invadopodia로 인한 ECM 저하의&gt; 시각화는 매트릭스 proteolysis 및 셀룰러 침해 가능성 4,5의 정도를 평가하기위한 가장 널리 기술로 떠오르고있다. 여기에 언급 된 상업적으로 이용 가능한 오레곤 그린 - 488 젤라틴의 공액을 활용 휘황 라벨이 표시된 유리 coverslips을 생성하기위한 표준 방법의 버전입니다.이 방법은 쉽게 빠르게 코팅 coverslips 많은 수의 생산 조정됩니다. 우리는 흔히 발생하는 일반적인 미세 유물의 일부를 보여 이 절차를 수행하는 동안 얼마나 이것들은 방지 할 수 있습니다. 마지막으로, 우리는 개별 셀 의해 전체 휴대폰 인구에 의해 중재 표시 젤라틴 매트릭스 저하의 정량화 할 수 있도록 즉시 사용할 수 컴퓨터 소프트웨어를 사용하여 표준화 된 방법을 설명합니다. 설명 절차는 정확하고 reproducibly 모니터링 할 수있는 기능을 제공 invadopodia활동, 또한 변조 단백질의 발현 또는 단일 및 다세포 설정에서 세포 외 기질 저하에 반 침해 화합물의 테스트의 효능을 평가하기위한 플랫폼으로 제공 할 수 있습니다.

Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

우리는 invadopodia로 인한 매트릭스 저하를 시각화를위한 형광 젤라틴으로 코팅 현미경 coverslips를 생산을위한 원형 방법을 설명합니다. 사용 가능한 소프트웨어를 사용하여 전산 기술이 혼합 된 인구 내에 전체 미세 필드를 포함한 다세포 그룹에 대한 하나의 세포 매트릭스 proteolysis의 결과 수준을 정량화를 위해 제공됩니다.

단일 및 다세포 상황에서 Invadopodia로 인한 세포 외 기질 Proteolysis의 양적 측정

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

quantitative-measurement-invadopodia-mediated-extracellular-matrix

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19.1K 조회수.  West Virginia University. 우리는 invadopodia로 인한 매트릭스 저하를 시각화를위한 형광 젤라틴으로 코팅 현미경 coverslips를 생산을위한 원형 방법을 설명합니다. 사용 가능한 소프트웨어를 사용하여 전산 기술이 혼합 된 인구 내에 전체 미세 필드를 포함한 다세포 그룹에 대한 하나의 세포 매트릭스 proteolysis의 결과 수준을 정량화를 위해 제공됩니다.

비디오: Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

전체 세포 인구의 단일 셀 수준에서 Cyclin B의 Proteolysis 유학

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

연구

생물학

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 ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

프로브 기반의 실시간 PCR은 마이크로 RNA의 정량 측정을위한 접근 방법

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

이온 선택적 자체 참조 미세 전극 기술을 이용하여 세포 외 이온 플럭스 측정

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

질량 분석기 분석을위한 펩티드로 조직과 소화에서 세포 외 기질 단백질의 농축

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

효율적인 포유 동물 세포 발현 및 결정화에 대한 세포 외 당 단백질의 단일 단계 정화

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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 ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

침연 (macerated) 조직의 전자 현미경을 스캔하면 세포 외 매트릭스를 시각화하기

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

세포 유래 세포 외 매트릭스의 빠른, 분리, 기능 연구에 대한 확장 방법, 및 분석

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

마이크로 유체 장치를 사용하면 단일 신진 효모 세포의 수명과 세포 표현형을 측정하는

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...