Questo lavoro è stato finanziato dal Fonds de Recherche sur la Nature et les Technologies de Quebec, in Canada la Fondazione per l&#39;Innovazione e la Canadian Institutes of Health Research (CIHR).

1. Microfabbricazione della testa della sonda (processo non mostrato nel video) <ol><li> A Si 2 wafer, quattro pollici di diametro, 525 micron di spessore, con uno strato di 1 micron di spessore SiO2 termico è spincoated con photoresist (PR) per 45 s a 4000 giri. </li><li> Il wafer è precotti a 110C per 50 s, ed esposta attraverso una maschera con tutti gli elementi (porte e microcanali) per 5 s, sviluppato e sciacquato con DI. </li><li> La scoperta è inciso SiO2 via in una tamponata 01:07 acido fluoridrico (BHF) una soluzione ≈ 15 min (dewetting del substrato in cui SiO2 è stato inciso indica il completamento della etch). Un plasma O2 o acetone viene utilizzato in cenere o nastri il restante PR. </li><li> Un secondo strato PR è spin-rivestito a 1500 rpm per 45 s, ottenendo un overlayer spessore di ≈ 10 micron [31]. Il modello SiO2 sotto questo strato di PR è ancora visibile e viene utilizzato per allineare il wafer con una seconda maschera con le uniche porte. </li><li> Dopo l&#39;esposizione e lo sviluppo del PR, il wafer viene sciacquato, asciugato e postbaked a 95C per 20 min. </li><li> Il wafer di silicio è fissato su un wafer di supporto con fuso cera bianca per proteggere il mandrino. </li><li> Un plasma ad accoppiamento induttivo (ICP) DRIE è utilizzato per trasferire il PR ed embedded SiO2 modelli in topografia wafer in un processo in tre fasi: <ol><li> DRIE fare ≈ 500μmdeep porte in Si (modello definita dalla spessa PR). </li><li> Senza scaricare il wafer dalla macchina DRIE, il PR è incenerito con un plasma. </li><li> Il modello esposto SiO2 agisce come maschera per un secondo processo a secco etch, la creazione di 50 canali micron profondo, e aprendo il ripieno e le porte di ventilazione attraverso il wafer. Dopo lo scarico, il wafer supporto è staccata sotto un getto di acqua calda. Il wafer microlavorati viene pulito con acetone, etanolo e DI. </li></ol></li><li> Singoli chip MFP sono a dadini. </li><li> Un blocco di interfaccia PDMS è fabbricata la fusione in uno micromould composto da due strutturato poli (metilmetacrilato) (PMMA) elementi, una piastra in acciaio lucido che formano la parte inferiore, e due capillari (ciascuno inserito in una delle due vias accesso buchi nel lamiera di acciaio) che serve come segnaposto per i fori di collegamento fluidica. Il PDMS è curato in forno a 60 ° C per almeno 1 h. </li><li> Il blocco PDMS è legato ad un dadini chip di silicio MFP, attivando entrambe le parti in aria plasma a 1 mbar per 24 s a 230W, e unendo le due cose insieme con un home-made aiuto per l&#39;allineamento meccanico. </li><li> L&#39;assemblea è lasciato al bond in un forno a 60 ° C per un minimo di 1 ora </li></ol> 2. Assemblea della MFP <ol><li> Tenuta di gas siringhe di vetro sono riempiti con i reagenti appropriati con siringhe e aghi di plastica per garantire che non siano presenti bolle d&#39;aria. Tipicamente, si usa una siringa da 1 -10 microlitro per iniezione, e una siringa con un volume di 5-10 volte più grande per l&#39;aspirazione. </li><li> Le siringhe sono collegati a tubi capillari utilizzando raccordi Nanotight a basso volume morto. </li><li> Capillari sono riempiti e controllati per bolle sotto il microscopio. </li><li> Il chip MFP è pre-riempite con soluzione tampone per evitare cattura di bolla quando si collegano i capillari. </li><li> I capillari sono collegati al raccordo PDMS nella testa della sonda </li></ol> 3. Set-up della MFP <ol><li> La testa della sonda è bloccato nel supporto sonda e montato sulla sonda stazione in cima ad un microscopio invertito </li><li> Le siringhe sono posti in alta precisione pompe siringa. </li><li> Il substrato, come ad esempio un vetrino, viene inserito in una porta fatta in casa che viene apposta sul palco microscopio. </li><li> Il parallelismo della mesa del MFP e il substrato è regolato con un paio di goniometri osservando gli anelli di Newton (frange di interferenza) che appaiono quando la stampante MFP è portato a contatto con il substrato. Il punto di contatto e la frequenza degli anelli servono come indicazione del tilt. Quando la stampante MFP è allineato con la superficie, un anello di interferenza singolo si estende su tutta la superficie. Questa misura serve anche per calibrare la separazione tra MFP e substrato. </li><li> Il divario tra il MFP e il substrato è un fattore critico per i processi di patterning superficie. Perché il substrato è elaborato dalla scansione al di sotto del MFP, l&#39;allineamento orizzontale deve essere regolato con precisione micrometrica ed è realizzato utilizzando un supporto a tre punti formato da tre viti micrometriche. </li></ol> 4. Funzionamento del MFP <ol><li> Erogazione è controllata via software LabView. Funzionamento del dispositivo viene visualizzato ad occhio e utilizzando una telecamera CCD. L&#39;iniezione: rapporto aspirazione varia 1:03-1:10, a seconda della diffusività del reagente con il tampone circostante e il modello desiderato flusso geometrico. </li><li> Per controllare il corretto funzionamento della siringa di aspirazione e la presenza di bolle, prima iniettare liquido con la siringa di aspirazione prima di iniziare l&#39;aspirazione corretta.</li><li> Inizio iniezione di liquido e il flusso di monitorare e confinamento di perline o di colorante tracciante fluorescente. </li><li> Utilizzare la sonda per la particolare applicazione, cioè la scansione attraverso la superficie per la lavorazione per la deposizione, di attacco o macchie di superficie o di cellule. </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>

La sonda microfluidica (MFP) è versatile perché è (i) mobili, (ii) adattabile per essere utilizzato con diversi tipi di reagenti e di substrato e può (iii) essere utilizzato su grandi aree. Bolle indesiderate può portare alla interruzione del flusso per evitare bolle, tutti i componenti devono essere pieni di liquidi prima del montaggio. Il divario tra la sonda e la superficie è solo di pochi micrometri, ma il mesa è diverse centinaia di micron di larghezza, e distanze nel range di ce...

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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

Dispositivi microfluidici permettono test deve essere eseguito utilizzando piccole quantità di campione e sono stati recentemente utilizzati per controllare il microambiente delle cellule. Microfluidica è comunemente associato con microcanali chiuse che limitano il loro uso per campioni che possono essere introdotte, e colto nel caso delle cellule, all&#39;interno di un volume confinato. D&#39;altra parte, micropipetting sistema sono stati utilizzati per perfusione a livello locale le cellule e le superfici, in particolare con push-pull configurazioni in cui si agisce pipetta come sorgente e l&#39;altra come affondare, ma il confinamento del flusso è difficile in tre dimensioni. Inoltre, pipette sono fragili e difficili da posizione e, quindi, sono utilizzati in configurazione statica solo. La microfluidica sonda (MFP) elude i vincoli imposti dalla costruzione di chiuse e canali microfluidica invece che racchiude il campione nel sistema microfluidica, il flusso microfluidica può essere consegnato direttamente sul campione, e la scansione attraverso il campione, usando la MFP. . L&#39;iniezione e aperture di aspirazione si trovano a poche decine di micrometri l&#39;uno dall&#39;altro in modo che un microjet iniettato il divario è confinato dalle forze idrodinamiche del liquido circostante e interamente aspirato nuovamente dentro l&#39;altra apertura. La microjet può essere lavato tutta la superficie del substrato e fornisce uno strumento preciso per la deposizione localizzata / fornitura di reagenti che possono essere utilizzate su vaste aree mediante la scansione di sonda su tutta la superficie. In questo video vi presentiamo la microfluidica sonda 1 (MFP). Vi spieghiamo in dettaglio come assemblare il MFP, montarlo in cima a un microscopio invertito, e allinearlo rispetto alla superficie del substrato e, infine, mostrano come usarlo per elaborare una superficie substrato immerso in un buffer.

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 questo video vi presentiamo la sonda microfluidica<sup&gt; 1</sup&gt; (MFP). Vi spieghiamo in dettaglio come assemblare il MFP, montarlo in cima a un microscopio invertito, e allinearlo rispetto alla superficie del substrato e, infine, mostrano come usarlo per elaborare una superficie substrato immerso in una soluzione tampone.

La sonda Microfluidic: funzionamento e utilizzo per l&#39;elaborazione delle superfici localizzate

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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Biology

8.8K Views.  McGill University. In questo video vi presentiamo la sonda microfluidica 1 (MFP). Vi spieghiamo in dettaglio come assemblare il MFP, montarlo in cima a un microscopio invertito, e allinearlo rispetto alla superficie del substrato e, infine, mostrano come usarlo per elaborare una superficie substrato immerso in una soluzione tampone.

Video: 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.

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

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 ...

Ricerca

Biologia

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.

Realizzazione e di funzionamento di un Inserto di ossigeno per colture cellulari aderenti

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.

Saggi biofisici per sondare le proprietà meccaniche del nucleo della cellula Interphase: Strain supporto Applicazione e manipolazione microago

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.

Avidità-based Screening Interaction extracellulare (AVEXIS) per il rilevamento scalabile di bassa affinità extracellulare del recettore-ligando interazioni

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.

L&#39;uso di chemostati in Biologia microbica Sistemi

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.

Iniezione intraduttale per Localizzato Drug Delivery per il mouse ghiandola mammaria

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.

Base-Probe Real-time PCR Approcci per la misurazione quantitativa dei microRNA

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.

Macchie di sangue essiccato - preparazione ed elaborazione per l'uso in test immunologici e tecniche molecolari

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.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

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.

Legatura del dotto biliare parziale nel topo: un modello radiocomandato di colestasi ostruttiva localizzata

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