Diese Arbeit wurde vom Fonds de Recherche sur la Nature et les Technologies de Quebec, Canada die Fundation für Innovation und die Canadian Institutes of Health Research (CIHR) finanziert.

1. Microfabrication des Tastkopfes (Prozess nicht in dem Video zu sehen) <ol><li> Ein Si 2-Wafer, vier Zentimeter im Durchmesser, 525 um dick, mit einer 1 um dicken thermischen SiO2-Schicht mit einem Photoresist (PR) für 45 s bei 4000 rpm aufgeschleudert. </li><li> Der Wafer wird bei 110C für 50 s vorgebackenen und ausgesetzt durch eine Maske mit allen Elementen (Häfen und Mikrokanäle) für 5 s, entwickelt und gespült, DI. </li><li> Die freigelegten SiO2 liegt versteckt in einer 1.07 geätzt gepufferter Flusssäure (BHF)-Lösung in ≈ 15 min (Entnetzung des Substrats, wo SiO2 geätzt wurde zeigt Abschluss der Etch). Ein O2-Plasma oder Aceton wird zu Asche verwendet oder Streifen die restlichen PR. </li><li> Ein zweiter PR-Schicht ist durch Spin-Coating bei 1500 rpm für 45 s, was eine dicke Deckschicht von ≈ 10 pm [31]. Die SiO2-Muster unter diesem PR-Schicht ist noch sichtbar und wird verwendet, um den Wafer mit einer zweiten Maske mit den Häfen nur auszurichten. </li><li> Nach der Belichtung und Entwicklung des PR wird der Wafer gespült, getrocknet und postbaked bei 95C für 20 min. </li><li> Die Si-Wafer wird auf einen Träger-Wafer mit geschmolzener weißer Wachs auf das Futter zu schützen befestigt. </li><li> Ein induktiv gekoppeltem Plasma (ICP) DRIE wird verwendet, um die PR übertragen und SiO2-Muster eingebettet in Wafer-Topographie in einem dreistufigen Prozess: <ol><li> DRIE auf ≈ 500μmdeep Ports in Si (Muster, das durch die dicken PR definiert) zu machen. </li><li> Ohne Entladen der Wafer aus der DRIE Maschine, ist die PR verascht mit einem Plasma. </li><li> Die freiliegenden SiO2-Muster dient als Maske für eine zweite Trockenätzprozess, wodurch 50 um tiefe Kanäle, und die Öffnung der Befüllung und Entlüftung Ports durch den Wafer. Nach dem Entladen wird die Unterstützung Wafer unter einem Strom von warmem Wasser gelöst. Der mikromechanische Wafer wird dann mit Aceton, Ethanol und DI gereinigt. </li></ol></li><li> Individuelle MFP-Chips werden in Würfel geschnitten. </li><li> Ein PDMS-Interface-Block wird durch Gießen in eine micromould von zwei strukturierten Poly (Methylmethacrylat) (PMMA)-Elemente, eine polierte Stahlplatte bildet den Boden, und zwei Kapillaren (jeweils in einer der beiden Vias-Zugang eingeführt Löcher-in den zusammengesetzten hergestellt Stahlplatte) dienen als Platzhalter für die fluidische Verbindung Löcher. Das PDMS wird in einem Ofen bei 60 ° C für mindestens 1 h ausgehärtet </li><li> Die PDMS-Block ist es, eine gewürfelte MFP Silizium-Chip durch die Aktivierung beider Teile in Luft Plasma bei 1 mbar für 24 s bei 230W und Zusammenfügen der beiden zusammen mit einem hausgemachten mechanische Ausrichthilfe verklebt. </li><li> Die Montage ist auf Bindung in einem 60C Ofen für mindestens 1 Stunde links </li></ol> 2. Versammlung der MFP <ol><li> Gasdichte Glasspritzen sind mit den entsprechenden Reagenzien mit Kunststoff-Spritzen und Nadeln, um sicherzustellen, dass keine Luftblasen gefüllt sind. Normalerweise verwenden wir einen 1 -10 Mikroliterspritze zur Injektion, und eine Spritze mit 5-10 mal größeres Volumen für Aspiration. </li><li> Die Spritzen sind mit den Schläuchen Kapillare mit Nanotight Armaturen mit geringem Totvolumen. </li><li> Kapillaren sind gefüllt und geprüft Blasen unter dem Mikroskop. </li><li> Die MFP-Chip ist mit Pufferlösung zu verhindern Einfangen von Blasen beim Anschließen der Kapillaren vorausgefüllt. </li><li> Die Kapillaren sind in die PDMS Stutzen gesteckt im Sondenkopf </li></ol> 3. Set-up des MFP <ol><li> Der Messkopf wird in die Sonde Halterung eingespannt und befestigt an der Sonde Station oben auf einem inversen Mikroskop </li><li> Die Spritzen werden in hochpräzisen Spritzenpumpen platziert. </li><li> Das Substrat, wie z. B. eine Glasplatte, in eine hausgemachte Halter, die auf dem Mikroskoptisch angebracht wird eingefügt. </li><li> Die Parallelität der mesa des MFP und das Substrat wird mit Hilfe eines Paares von Goniometer durch Beobachtung der Newtonschen Ringe (Interferenzstreifen), wenn der MFP ist in Kontakt mit dem Substrat gebracht erscheinen. Die Anlaufstelle und die Häufigkeit der Ringe dienen als Indiz für die Neigung. Wenn der MFP mit der Oberfläche ausgerichtet ist, erstreckt sich eine einzelne Störung Ring über die gesamte Fläche. Auch diese Maßnahme dient dazu, die Trennung zwischen MFP und Substrat zu kalibrieren. </li><li> Die Kluft zwischen dem MFP und das Substrat ist von entscheidender Bedeutung für die Oberflächenstrukturierung Prozesse. Da das Substrat durch Scannen sie unter dem MFP verarbeitet wird, hat die horizontale Ausrichtung, mit Mikrometer-Präzision eingestellt werden und wird durch eine Drei-Punkt-Unterstützung durch drei Mikrometerschrauben gebildet. </li></ol> 4. Der Betrieb des MFP <ol><li> Dispensing über LabView-Software gesteuert. Bedienung des Gerätes wird visualisiert durch Auge und mit einer CCD-Kamera. Die Injektion: aspiration Verhältnis variiert von 1.03 bis 01.10 Uhr, abhängig von der Diffusivität des Reagenzes mit der umgebenden Puffer und der gewünschten geometrischen Strömungsmuster. </li><li> Um zu überprüfen, für den ordnungsgemäßen Betrieb der Aspirationsspritze und das Vorhandensein von Blasen, zuerst spritzen Flüssigkeit mit dem Aspirationsspritze bevor richtigen Aspiration.</li><li> Start Einspritzen von flüssigen und Monitor fließen und der Entbindung von Perlen oder fluoreszierender Tracer-Farbstoff. </li><li> Verwenden Sie die Sonde für die jeweilige Anwendung, dh über Oberflächen-Scan für die Verarbeitung für die Abscheidung, Ätzen oder Beizen der Oberfläche oder Zellen. </li></ol>

<ol>
	<li>Juncker, D., Schmid, H., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature Materials. 4 (8), 622-622 (2005).</li></ol>

Die mikrofluidischen Sonde (MFP) ist vielseitig, weil es (i) mobil ist, (ii) zur Verwendung mit verschiedenen Arten von Reagenzien und Substrat, und es kann (iii) über große Flächen betrieben werden. Unerwünschte Blasen können zu einer Störung der Strömung, um Blasen zu vermeiden, führen, müssen alle Komponenten mit Flüssigkeiten vor der Montage befüllt werden. Die Kluft zwischen der Sonde und die Oberfläche ist nur wenige Mikrometer, noch die Mesa ist mehrere hundert Mikrometer ...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>microfluidic connectors</td>
<td>&nbsp;</td>
<td>Upchurch Scientific</td>
<td>Micro- and Nano-tight fittings and sleeves</td>
<td>&nbsp;</td>
<tr>
<td>2-component manual dispenser</td>
<td>&nbsp;</td>
<td>Conprotec Inc.</td>
<td>DM400</td>
<td>To dispense and mix PDMS mixture</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Version 8.0</td>
<td>&nbsp;</td>
<tr>
<td>Mechanical Convection Oven</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>1330FM</td>
<td>&nbsp;</td>
<tr>
<td>Glass syringes</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubing</td>
<td>&nbsp;</td>
<td>Polymicro Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasma Chamber</td>
<td>&nbsp;</td>
<td>Tegal Corporation</td>
<td>Plasmaline 415</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>TE2000-E</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pumps</td>
<td>&nbsp;</td>
<td>Cetoni</td>
<td>neMESYS</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Ellsworth Adhesives</td>
<td>184 Sil Elast Kit</td>
<td>&nbsp;</td>
<tr>
<td>Camera</td>
<td>&nbsp;</td>
<td>Photometrics</td>
<td>QuantEM 512SC</td>
<td>&nbsp;</td>
<tr>
<td>Microscope stage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microfluidic probe holder goniometers</td>
<td>&nbsp;</td>
<td>Melles-Griot</td>
<td>07GON504</td>
<td>&nbsp;</td>
<tr>
<td>Linear stage</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>LS-50</td>
<td>For z-control of the MFP</td>
</tr>
<tr>
<td>Manual linear stage</td>
<td>&nbsp;</td>
<td>Newport</td>
<td>443-4 Series</td>
<td>For x- and y- axis control of the MFP</td>
</tr>
<tr>
<td>Microscope stage</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>PZ-2000</td>
<td>With x-, y- and z- control</td>
</tr>		</tbody>
		</table>
		</div>

the microfluidic probe: operation and use for localized surface processing

Mikrofluidik-Geräte ermöglichen die Tests durchgeführt mit winzigen Mengen der Probe werden und haben vor kurzem benutzt worden, um die Mikroumgebung der Zellen steuern. Die Mikrofluidik ist häufig mit geschlossenen Mikrokanälen, die ihren Einsatz einschränken, um Proben, die eingeführt werden, und können kultiviert im Falle von Zellen innerhalb eines begrenzten Volumens verbunden. Auf der anderen Seite haben Mikropipettiersystem verwendet worden, um vor Ort perfuse Zellen und Oberflächen, vor allem mit Push-Pull-Setups, wo eine Pipette dient als Quelle und der andere als sinken, aber die Beschränkung der Strömung wird in drei Dimensionen schwierig. Darüber hinaus sind Pipetten zerbrechlich und schwierig zu positionieren und damit in eine statische Konfiguration nur verwendet. Die mikrofluidischen Sonde (MFP) umgeht die Einschränkungen durch den Bau von verschlossenen mikrofluidischen Kanälen verhängt und statt der Beifügung der Probe in die Mikrofluidik-System, der mikrofluidischen Durchfluss kann direkt auf die Probe geliefert werden, und gescannte über die Probe, mit dem MFP. . Die Injektion und Ansaugöffnungen sind in wenigen zehn Mikrometern voneinander entfernt, so dass ein microjet in den Spalt injiziert durch die hydrodynamischen Kräfte der umgebenden Flüssigkeit beschränkt ist und ganz wieder in die andere Öffnung abgesaugt. Die microjet kann über die Substratoberfläche gespült werden und liefert ein präzises Werkzeug für lokalisierte Ablagerung / Lieferung von Reagenzien, die über große Flächen durch das Scannen der Sonde auf der Oberfläche verwendet werden können. In diesem Video stellen wir Ihnen die mikrofluidischen Sonde 1 (MFP). Wir erklären im Detail, wie Sie die MFP montieren, montieren Sie ihn oben auf einem inversen Mikroskop, und richten Sie sie in Bezug auf die Substratoberfläche und schließlich zeigen, wie man es verwenden, um eine Substratoberfläche in einem Puffer eingetaucht Prozess.

Watch this Scientific Journal Video about The Microfluidic Probe: Operation and Use for Localized Surface Processing at JoVE.com

The Microfluidic Probe: Operation and Use for Localized Surface Processing

In diesem Video stellen wir Ihnen die mikrofluidischen Sonde<sup&gt; 1</sup&gt; (MFP). Wir erklären im Detail, wie Sie die MFP montieren, montieren Sie ihn oben auf einem inversen Mikroskop, und richten Sie sie in Bezug auf die Substratoberfläche und schließlich zeigen, wie man es verwenden, um eine Substratoberfläche in einer Pufferlösung eingetaucht Prozess.

Die Mikrofluidik-Probe: Betrieb und Nutzung für lokalisierte Oberflächenbearbeitung

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

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JoVE Journal

Biology

8.8K Aufrufe.  McGill University. In diesem Video stellen wir Ihnen die mikrofluidischen Sonde 1 (MFP). Wir erklären im Detail, wie Sie die MFP montieren, montieren Sie ihn oben auf einem inversen Mikroskop, und richten Sie sie in Bezug auf die Substratoberfläche und schließlich zeigen, wie man es verwenden, um eine Substratoberfläche in einer Pufferlösung eingetaucht Prozess.

Serie: The Microfluidic Probe: Operation and Use for Localized Surface Processing

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Lokalisierte RNAi und ektopische Genexpression in der Blutegel

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Forschung

Biologie

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Herstellung und Betrieb eines Oxygen Insert für adhärente Zellkulturen

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Biophysikalische Assays auf die mechanischen Eigenschaften der Interphase Zellkern Probe: Substrat Dehnungs-Anwendung und Mikronadel Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidität-basierte extrazelluläre Wechselwirkung Screening (AVEXIS) für die Erkennung von Scalable Low-Affinität extrazellulären Rezeptor-Ligand-Wechselwirkungen

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Intraduktale Injection für Lokalisierte Drug Delivery auf die Maus Milchdrüse

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

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.

Sonde-basierte Echtzeit-PCR Ansätze zur quantitativen Messung von 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 ...

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e., collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.

The preparing and processing of dried blood spots (DBS) for their final analysis are still poorly standardized for most diagnostic applications. To overcome this shortcoming, a comprehensive step-by-step protocol is suggested and subsequently evaluated with regard to its effectiveness for detecting markers of viral infections.

Getrocknete Blutflecken - Vorbereitung und Verarbeitung für den Einsatz in Immunoassays und Molekulare Techniken

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

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.

Mikrofluidik-Geräte mit Measure Lebensdauer und Cellular Phänotypen in Einzel Budding Hefezellen

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

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

Partielle Gallengang Ligation in der Maus: eine kontrollierte Modell der lokalisierten obstruktive Cholestase

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...