I canali microfluidica sono fabbricati in poliolefine ciclico (Zeonex ® 690R, Zeon Chemical Inc., Louisville, KY). Zeonex ha una temperatura di transizione vetrosa (Tg) di 136 ° C ed è trasparente ai raggi UV, che è essenziale per la chimica luce diretta utilizzato nella formazione porosa monolite. I microcanali sono realizzati da micro-hot-embossing con Nico elettroformati master-stampo, che è il negativo (inverso) caratteristiche della piattaforma microfluidica. Il caldo, goffratura è fatto come segue: <ol><li> Lo stampo e il substrato polimerico sono posti tra due piastre metalliche parallele. </li><li> Le piastre vengono riscaldate fino alla temperatura di rilievo (166 ° C), che è di 30 º C al di sopra della T g di Zeonex. </li><li> Le piastre vengono premuti insieme per circa 5 min e la pressione è mantenuta a 250 psi. </li><li> Lo stampo e il substrato vengono poi rimossi dai piani riscaldati, lasciato raffreddare per 1-2 minuti, e poi manualmente separati l&#39;uno dall&#39;altro. </li><li> Pozzi di 1,5 mm di diametro sono forati alle estremità dei canali per l&#39;introduzione del campione e la raccolta. </li><li> Il substrato di plastica in rilievo è poi termosaldata con un altro pezzo di Zeonex al modulo allegato canali microfluidica. Il substrato in rilievo e il pezzo di copertura può essere UV-ozono o plasma trattati prima dell&#39;incollaggio termico per migliorare l&#39;efficienza di tenuta e garantire la fedeltà della struttura del canale chiuso (Bhattacharyya e Klapperich, 2007). </li></ol> Fabbricazione di estrazione in fase solida (SPE) colonna all&#39;interno dei canali di plastica microfluidica Realizzazione della fase solida è un processo in due fasi. In un primo momento, i microcanali fabbricato sono superficie modificata in modo da migliorare l&#39;adesione della colonna SPE al dispositivo in plastica. L&#39;innesto tramite fotopolimerizzazione superficie viene utilizzato per funzionalizzare le pareti della microcanali polimerici. La procedura di innesto è la seguente: <ol><li> I microcanali sono riempite con una miscela 1:1 di diacrilato etilene (EDA) e metil metacrilato (MMA) con 3% benzofenone, che è un fotoiniziatore idrogeno astrazione. L&#39;, EDA MMA e benzofenone sono acquistati da Sigma-Aldrich, St. Louis, MO. </li><li> Il chip viene poi UV irradiata per 10 minuti a 254 nm di lunghezza d&#39;onda UV e 200 mJ / cm 2 di energia in uno strumento esposizione ai raggi ultravioletti (CL-1000 Crosslinker UV, UPV Inc., Ontario, CA). L&#39;innesto avviene attraverso H-astrazione polimerizzazione e dei risultati in superficie legato catene di polimeri, che vengono inseriti all&#39;interno di polimerizzazione la miscela utilizzata per la preparazione del monolite nel secondo passaggio. </li><li> Il monomero in eccesso viene rimosso dai canali di risciacquo con 15 ml di metanolo con l&#39;aspirazione a vuoto. </li></ol> Dopo il processo di photografting, un polimero microporoso monolite è costituito all&#39;interno della microcanali che intrappolano le particelle di silice per l&#39;estrazione del DNA. Il monolite poroso viene preparato in situ come segue: <ol><li> I canali superficie modificata sono riempite con una miscela precursore monolite composto da Buma (15% in peso), EDMA (10% in peso), 1-dodecanolo (52,5% in peso), cicloesanolo (22,5% in peso), DMPAP (peso 1% rispetto di monomeri) e 0,7 micron particelle di silice (15% rispetto al volume totale del pre-polimero misto). Le microsfere di silice sono stati acquistati da Polysciences, Inc. (Warrington, PA) ed i monomeri e solventi porogenic sono acquistati da Sigma-Aldrich, St. Louis, MO. </li><li> Il chip viene irradiato con raggi UV nel reticolante UV a 200 mJ / cm 2 per 1.1 minuti per iniziare la polimerizzazione, e il monomero in eccesso viene rimosso dai canali di risciacquo con 15 ml di metanolo con l&#39;aspirazione a vuoto. </li><li> I collegamenti fluidica (NanoPorts connessioni TM, Upchurch scientifico, Oak Harbor, WA) sono poi attaccati l&#39;introduzione e pozzetti di raccolta dei canali microfluidica. </li><li> Il monolite poroso viene poi lavato di nuovo con il metanolo per almeno 30 minuti utilizzando la pompa a siringa con un flusso di 100mL/hour. </li></ol> Fabbricazione di CNT lisi monolite all&#39;interno dei canali di plastica microfluidica Realizzazione della (nanotubi di carbonio) lisi monolite CNT è un processo in due fasi. In primo luogo, i microcanali fabbricato sono superficie modificata (innestata) nello stesso modo come per le colonne SPE nel dispositivo di plastica. Dopo il processo di photografting, un monolite microporoso polimero si forma all&#39;interno del microcanali che è impregnato di multi-parete nanotubi di carbonio. Il monolite poroso viene preparato in situ come segue: <ol><li> Multi-nanotubi a parete sono risospesi in cicloesanolo ad una concentrazione di 2.27M. Questo risospensione è ultrasonicated per 30 minuti al 50% di ampiezza e duty cycle del 50% con una cella sonifier distruttore (Sonifier S-250D, prodotto da ultrasuoni Branson, Danbury, CT). La sonicazione fornito una sospensione efficace e stabile 0.5M di CNT. I nanotubi di carbonio sono stati acquistati da Nanolabs (Brighton, MA). </li><li> I canali superficie modificata sono riempiti con una miscela simile precursore monolite solo che adesso la soluzione di cicloesanolo + CNT viene utilizzato. La miscela monolite è costituito da Buma (15% in peso), EDMA (10% in peso), 1-dodecanolo (52,5% in peso), e cicloesanolo + nanotubi di carbonio (22,5% in peso). Per questa soluzione il DMPAP fotoiniziatore viene aggiunto (1,13% in peso rispetto ai monomeri). I monomeri, solventi e porogenic DMPAPA sono acquistati da Sigma-Aldrich, St. Louis, MO. </li><li> Il chip viene irradiato con raggi UV nel reticolante UV a 120 mJ / cm 2 per 0,9 minuti per iniziare la polimerizzazione. Il dispositivo viene capovolto di 180 gradi in crossliker e irradiati per un altro min 0,9 a 120 mJ / cm 2. Il monomero in eccesso viene rimosso dai canali di risciacquo con 50 ml di metanolo con l&#39;aspirazione a vuoto. </li><li> I collegamenti fluidica (NanoPorts connessioni TM, Upchurch scientifico, Oak Harbor, WA) sono poi attaccati l&#39;introduzione e pozzetti di raccolta dei canali microfluidica. </li></ol> Preparazione del campione batterico per l&#39;uso con CNT lisi monolito La preparazione del campione per un campione batterica consiste nel prelievo di una misurazione della densità ottica a 600nm per garantire che il campione non è estremamente concentrata in modo da evitare di intasare il canale. <ol><li> Prendete una lettura OD a 600nm con i batteri in media, questo è fatto con un BioPhotometer (Eppendorf BioPhotometer, Eppendorf Scientific, Inc., Westbury, NY). Diluire il campione fino a quando le letture OD rientrano nell&#39;intervallo 0,18-0,25 A. </li><li> Centrifugare il campione a 6.500 giri per 5 minuti e decantare il surnatante. </li></ol> Lisi <ol><li> Batteri risospendere in GuSCN * + 0,01% SDS + 4% proteinasi K (0.8mg/ml). Vortex brevemente (1 ml di batteri in media dovrebbero essere risospeso in 1ml GuSCN). Proteinasi K è stato aggiunto alla soluzione di un aiuto con denaturazione delle proteine ​​che sono attaccati al DNA batterico e di inibire DNAases e RNAases. Un ulteriore vantaggio della proteinasi K è che si aiuta anche con la rottura verso il basso le proteine ​​della parete cellulare, che è auspicabile in quanto entrambi i batteri gram-positivi e gram-negativi pareti cellulari dei batteri contengono grandi quantità di proteine ​​(anche se i batteri gram-negativi hanno in genere più proteine ). Il buffer caotropico aiuti ulteriormente la proteinasi K con denaturazione delle proteine, mentre il detergente rompe la parete cellulare. Proteinasi K e tiocianato guanidinio (GuSCN) sono stati acquistati da Qiagen Inc. (Valencia, CA). SDS (Dodecyl solfato di sodio) è stato acquistato da Pierce (Rockford, IL). </li><li> Immediatamente carico il campione in una siringa collegata al tubo di PEEK. Aspirare la siringa in modo che l&#39;aria non è nel sistema. Collegare il tubo alla nanoport e canale. </li></ol> Una pompa a siringa KDS100 (prodotto da KD scientifico, Holliston, MA) è stato utilizzato per gli esperimenti e la portata utilizzata è di 450 microlitri / ora per tutti i passaggi. Flusso del canale lisi campione a 450ul/hr. <ol><li> Raccogliere il campione e procedere con l&#39;estrazione del DNA tramite SPE (vedi protocollo di estrazione del DNA). Nota: questo esempio batteri non avrà bisogno di seguire passo carico 2, dal momento che la sospensione è GuSCN *. </li></ol> Protocollo di estrazione del DNA La procedura di estrazione è costituito da 3 fasi: <ol><li> Carico. </li><li> Washington </li><li> Eluire. </li></ol> Pompa a siringa KDS100 (prodotto da KD scientifico, Holliston, MA) è stato utilizzato per gli esperimenti e la portata è stato utilizzato 300 ml / h per tutti i passaggi. Carico <ol><li> Condizione il canale con il tampone caotropico (GuSCN *, guanidinio tiocianato) per 1-2 minuti prima di iniziare l&#39;esperimento. </li><li> Mescolare il campione con il tampone caotropico in rapporto 1:1. </li><li> Flusso di miscela campione attraverso il canale per 3 min. </li></ol> Lavare <ol><li> Lavare il canale con etanolo al 70% per 2 minuti. </li><li> Lavare il canale con il 100% di etanolo per 2 min. </li></ol> Eluire <ol><li> Eluire il DNA estratto in acqua Millipore per 3 min. Raccogliere 15 ml di purificato, PCR-ready DNA. </li></ol> * Il GuSCN dal kit Qiagen (buffer RLT) è stato utilizzato.

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

Nel nostro laboratorio, abbiamo isolato con successo gli acidi nucleici direttamente dai volumi e submicroliter microlitri di sangue umano, urine e feci con polimero / nanoparticelle lisi composito microscala e solide colonne di estrazione di fase. I campioni recuperati sono concentrati, i campioni di piccolo volume che sono PCRable, senza alcuna pulizia supplementare. Qui, dimostriamo come fabbricare chip termoplastico microfluidica con goffratura a caldo e saldatura a caldo. Poi, ci dimostrano come l&#39;uso in superficie chiara situ l&#39;innesto diretto e la polimerizzazione attraverso il chip sigillati a formare le colonne composte in fase solida. Dimostriamo innesto e polimerizzazione di una colonna di nanotubi di carbonio / polimero composito per lisi cellulare batterica. Poi mostrano il processo di lisi seguita da estrazione in fase solida degli acidi nucleici dal campione sul chip di silicio con un / colonna polimero composito. I protocolli allegati contengono istruzioni dettagliate su come fare sia lisi e colonne di estrazione in fase solida.

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

Qui mostriamo come fabbricare chip microfluidici termoplastico con goffratura a caldo e saldatura a caldo. Poi ci dimostrano come l&#39;uso in superficie chiara situ l&#39;innesto diretto e la polimerizzazione attraverso il chip sigillati a formare le colonne composte in fase solida.

Fabbricazione dei canali termoplastico Microfluidic

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

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K Views.  Boston University. Qui mostriamo come fabbricare chip microfluidici termoplastico con goffratura a caldo e saldatura a caldo. Poi ci dimostrano come l'uso in superficie chiara situ l'innesto diretto e la polimerizzazione attraverso il chip sigillati a formare le colonne composte in fase solida.

Video: Fabrication of the Thermoplastic Microfluidic Channels

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Ricerca

Biologia

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

Non plasmatici Incollaggio di PDMS Economico per la Fabbricazione di dispositivi microfluidici

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

Le domande Microfluidic per la diagnostica e getta

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Patch di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

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.

Fabbricazione di elettrodi amperometrici

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].

Fabbricazione di miogenici Costrutti di ingegneria tessutale

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

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomica per identificare proteine ​​che interagiscono con P2X2 Ligand-Gated canali di comunicazione

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

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. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...