ML wird von der NIA (R00AG033176 und R01AG040081) und Laboratory Directed Forschung und Entwicklung, US Department of Energy contract # DE-AC02-05CH11231 unterstützt.

<ol>
	<li>Bissell, M. J., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape,+cytoskeletal+tension,+and+RhoA+regulate+stem+cell+lineage+commitment.">Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.</a> Dev. Cell. 6, 483-495 (2004).</li><li>LaBarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+progenitor+cell+fate+decsions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decsions are products of interactions with combinatorial microenvironments.</a> Integrative Biology. 1, 70-79 (2009).</li><li>Kim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Methods+for+Polymers+in+Cell+and+Tissue+Engineering.">Patterning Methods for Polymers in Cell and Tissue Engineering.</a> Annals of biomedical engineering. , (2012).</li><li>Boudou, T., Ohayon, J., Picart, C., Pettigrew, R. I., Tracqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+elastic+properties+of+polyacrylamide+gels:+implications+for+quantification+of+cellular+forces.">Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces.</a> Biorheology. 46, 191-205 (2009).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current protocols in cell biology. Chapter 10, Unit 10 (2010).</li><li>Flaim, C. J., Chien, S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+matrix+microarray+for+probing+cellular+differentiation.">An extracellular matrix microarray for probing cellular differentiation.</a> Nat. Methods. 2, 119-125 (2005).</li><li>Soen, Y., Mori, A., Palmer, T. D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+regulation+of+human+neural+precursor+cell+differentiation+using+arrays+of+signaling+microenvironments.">Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.</a> Mol. Syst. Biol. 2, 37 (2006).</li></ol>

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Die MEArray hier vorgestellte Methode ermöglicht funktionelle Analysen von Zell-und kombinatorischen Mikroumgebung Interaktionen 4. MEArray Analyse kombiniert Verwendung von basischen Mikrostrukturierung Technologien, Zellbiologie, und Microarray-Druck Roboter und Analyse-Geräte, die in vielen Multiuser-Labore sind. MEArray Bildschirme sind kompatibel mit den meisten adhärente Zelltypen, wenn serumfreie Medien Formulierungen müssen möglicherweise in einigen Fällen eingestellt werden zu BSA oder &lt;1% S...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalognummer </td><td> Kommentare (optional) </td></tr><tr><td> Glasplättchen 25 mm x 75 mm </td><td> VWR </td><td> 48311-600 </td><td></td></tr><tr><td> Deckgläschen (Nr.1) 24 mm x 50 mm </td><td> VWR </td><td> 48393-241 </td><td></td></tr><tr><td> Färbekasten (oder Coplan jar) </td><td> VWR </td><td> 25461-003 </td><td></td></tr><tr><td> Petrischalen (15 cm) </td><td> BD Falcon </td><td> 351058 </td><td></td></tr><tr><td> NaOH (1,0 N) </td><td> Sigma-Aldrich </td><td> S2567 </td><td></td></tr><tr><td> APES (&gt; 98% (3-Aminopropyl) triethoxysilan) </td><td> Sigma-Aldrich </td><td> A3648 </td><td></td></tr><tr><td> Glutaraldehyd </td><td> Sigma-Aldrich </td><td> G7651 </td><td> 50% in Wasser </td></tr><tr><td> APS (&gt; 98% Ammoniumpersulfat) </td><td> Sigma-Aldrich </td><td> A3678 </td><td> Bereiten Sie 10% funktionierende Lösung mit ddH 2 O </td></tr><tr><td> TEMED (N, N, N &#39;, N&#39;-Tetramethylethylendiamin) </td><td> Sigma-Aldrich </td><td> T9281 </td><td></td></tr><tr><td> Acrylamid (40%) </td><td> Sigma-Aldrich </td><td> A4058 </td><td></td></tr><tr><td> Bis-Acrylamid (2% w / v) </td><td> Fisher BioReagents </td><td> BP1404-250 </td><td></td></tr><tr><td> 0,45 um Spritzenfilter 4-mm Nylon- </td><td> Nalgene </td><td> 176-0045 </td><td></td></tr><tr><td> FITC </td><td> Sigma-Aldrich </td><td> F4274 </td><td></td></tr><tr><td> PDMS (Polydimethylsiloxan) </td><td> Dow Corning </td><td> 3097358-1004 </td><td> Sylgard 184 Elastomer-Kit über Ellsworth Adhesives </td></tr><tr><td> 2-Kammer-Objektträgern </td><td> NUNC </td><td> 177380 </td><td></td></tr><tr><td> Pluronic F108 </td><td> BASF </td><td> 30089186 </td><td></td></tr><tr><td> Aquarium Dichtstoff </td><td> Dow Corning </td><td> DAP 00688 </td><td></td></tr><tr><td> Fluormount-G </td><td> Southern Biotech </td><td> 0100-01 </td><td></td></tr><tr><td> Einweg-Plastikbecher </td><td></td><td></td><td></td></tr><tr><td> Mundspatel </td><td></td><td></td><td></td></tr><tr><td> Nitril-Handschuhe </td><td></td><td></td><td></td></tr><tr><td> Plastic Objektträger-Boxen </td><td></td><td></td><td></td></tr><tr><td> Spin Coater </td><td> WS-400B-6NPP/LITE </td><td> Laurell Technologies Corporation </td><td></td></tr><tr><td> Ofen </td><td></td><td></td><td></td></tr><tr><td> Digitale Kochplatte </td><td></td><td></td><td></td></tr><tr><td> 384-Well-Platten </td><td></td><td></td><td> Eine Marke geeignet für die Mikroarray-Roboter </td></tr><tr><td> Microarray-Druck-Roboter </td><td></td><td></td><td></td></tr><tr><td> Inverted Phase und Fluoreszenz-Mikroskop </td><td></td><td></td><td></td></tr><tr><td> Axon Microarrayscannern </td><td> Molecular Devices </td><td></td><td> Mehrere Konfigurationen existieren </td></tr></tbody></table>

fabrication and use of microenvironment microarrays (mearrays)

Die Wechselwirkungen zwischen Zellen und ihrer Umgebung Mikroumgebung funktionelle Konsequenzen für zelluläres Verhalten. Auf der einzigen Zelle Ebene können unterschiedliche Mikroumgebungen verhängen Differenzierung, Migration und Proliferation Phänotypen, und auf der Gewebe-Ebene die Mikroumgebung verarbeitet so komplex wie Morphogenese und Tumorentstehung 1. Nicht nur, dass die Zell-und Molekularbiologie Inhalt Mikroumgebungen Auswirkungen die Zellen innerhalb, aber so tun die Elastizität 2 und Geometrie 3 des Gewebes. Definiert als die Summe der Zell-Zell-,-ECM und löslichen Faktor Wechselwirkungen neben physikalischen Eigenschaften ist der Mikroumgebung komplex. Die Phänotypen von Zellen innerhalb eines Gewebes sind teilweise auf Grund ihrer genomischen Inhalt und teilweise auf die Wechselwirkungen mit der kombinatorischen microenviroment. Eine große Herausforderung ist es, bestimmte Kombinationen von Mikroumgebung Komponenten mit charakteristischen Verhaltensweisen zu verknüpfen. ent &quot;&gt; Hier präsentieren wir die Mikroumgebung Mikroarray (MEArray)-Plattform für zellbasierte funktionelles Screening von Wechselwirkungen mit kombinatorischen Mikroumgebungen 4. Das Verfahren erlaubt die gleichzeitige Steuerung der molekularen Zusammensetzung und der Elastizitätsmodul und kombiniert die Verwendung von allgemein verfügbar Microarray und Mikrostrukturierung Technologien. MEArray Bildschirme benötigen so wenige wie 10.000 Zellen pro Array, welches funktionelle Studien von seltenen Zelltypen wie adulte Progenitorzellen erleichtert. Eine Begrenzung der Technik ist, dass ganze Gewebe Mikroumgebungen nicht vollständig auf MEArrays rekapituliert werden. jedoch Vergleich Antworten in der gleichen Zelltyp zu zahlreichen verwandten Mikroumgebungen beispielsweise paarweise Kombinationen von ECM-Proteinen, die eine gegebene Gewebes zu charakterisieren, werden Erkenntnisse, wie Mikroumgebungsbedingungen Komponenten hervorzurufen gewebespezifischen funktionellen Phänotypen bereitzustellen. MEArrays gedruckt unter Verwendung einer Vielzahl von rekombinantem gro werdenwth Faktoren, Cytokine und gereinigtes ECM Proteine, und Kombinationen davon. Die Plattform wird nur durch die Verfügbarkeit von spezifischen Reagenzien begrenzt. MEArrays zugänglich sind Zeitraffer Analyse, aber am häufigsten für Endpunkt-Analysen von Zellfunktionen, die messbar sind mit fluoreszierenden Sonden verwendet. Zum Beispiel werden DNA-Synthese, Apoptose, Akquisition von differenzierten Zustände oder Produktion spezifischer Genprodukte häufigsten gemessen. Kurz gesagt, ist der grundlegende Ablauf eines MEArray Experiment Folien mit Druck Substrate beschichtet vorzubereiten und die Master-Platte von Proteinen, die gedruckt werden sollen, vorbereitet. Dann werden die Arrays mit einem Mikroarray-Roboter gedruckt werden Zellen erlaubt zu befestigen, in Kultur zu wachsen, und werden dann chemisch bei Erreichen der experimentellen Endpunkt fixiert. Fluoreszierenden oder kolorimetrische Assays, mit traditionellen Mikroskopen oder Microarrayscannern abgebildet wird, werden verwendet, um relevante molekularen und zellulären Phänotypen (Abbildung 1) offenbaren.

Watch this Scientific Journal Video about Fabrication and Use of MicroEnvironment microArrays MEArrays at JoVE.com

Fabrication and Use of MicroEnvironment microArrays MEArrays

Eine kombinatorische funktionelle Screening-Methode für Einblicke in die Auswirkungen der molekularen Zusammensetzung der Mikroumgebung auf zelluläre Funktionen beschrieben. Das Verfahren nutzt bestehende Biochip-basierten Technologien, um Arrays von definierten kombinatorischen Mikroumgebungen, dass Zelladhäsion und funktionelle Analyse unterstützt generieren.

Herstellung und Verwendung von Mikroumgebung Microarrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

fabrication-and-use-of-microenvironment-microarrays-mearrays

Research

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Biology

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

9.9K Aufrufe.  Lawrence Berkeley National Laboratory. Eine kombinatorische funktionelle Screening-Methode für Einblicke in die Auswirkungen der molekularen Zusammensetzung der Mikroumgebung auf zelluläre Funktionen beschrieben. Das Verfahren nutzt bestehende Biochip-basierten Technologien, um Arrays von definierten kombinatorischen Mikroumgebungen, dass Zelladhäsion und funktionelle Analyse unterstützt generieren.

Serie: Fabrication and Use of MicroEnvironment microArrays MEArrays

Bacterial Gene Expression Analysis Using Microarrays

Bakterielle Gene Expression Analysis Using Microarrays

Forschung

Biologie

PDMS Device Fabrication and Surface Modification

PDMS Bauelementherstellung und Oberflächenmodifizierung

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

Die Herstellung der thermoplastischen Mikrofluidikkanälen

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Herstellung von amperometrische Elektroden

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Herstellung von Myogenic Engineered Tissue Konstrukte

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Genomweite Analyse der Aminoacylierung (Charging) Ebenen der tRNA mittels Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA Microarrays: Sample Quality Control, Array Hybridisierung und Scannen

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

Herstellung von Tissue Microarrays, Immunhistochemie Färbung und Digitalisierung Innerhalb des Human Protein Atlas

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Glycan Profiling of Plant Cell Wall Polymers mit Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Der Einsatz von Chemostate in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...