Les canaux microfluidiques sont fabriquées dans une polyoléfine cyclique (Zeonex ® 690R, Zeon Chemical Inc, Louisville, KY). Zeonex a une température de transition vitreuse (Tg) de 136 ° C et est transparente aux UV, ce qui est essentiel pour la chimie lumière dirigée utilisé dans la formation poreuse monolithe. Les microcanaux sont fabriqués par des micro-gaufrage à chaud avec un maître-NiCo électroformés moule qui a la borne négative (inverse) caractéristiques de la plate-forme microfluidique. Le gaufrage à chaud se fait comme suit: <ol><li> Le moule et le substrat polymère sont placées entre deux plaques métalliques parallèles. </li><li> Les plateaux sont chauffés jusqu&#39;à la température de gaufrage (166 ° C), ce qui est de 30 º C au-dessus du T g de Zeonex. </li><li> Les plateaux sont ensuite pressées pendant environ 5 min et la pression est maintenue à 250 psi. </li><li> Le moule et le substrat sont ensuite retirés des plateaux chauffés, on laisse refroidir pendant 1-2 min, puis manuellement séparés les uns des autres. </li><li> Des puits de 1,5 mm de diamètre sont percés aux extrémités des canaux pour l&#39;introduction de l&#39;échantillon et la collecte. </li><li> Le substrat en plastique gaufré est alors thermoliés avec un autre morceau de Zeonex au formulaire ci-joint canaux microfluidiques. Le substrat en relief et la pièce de couverture peut être UV-ozone ou plasma traité avant le collage thermique pour améliorer l&#39;efficacité de l&#39;étanchéité et s&#39;assurer de la fidélité de la structure du canal fermé (Bhattacharyya et Klapperich, 2007). </li></ol> Fabrication d&#39;extraction en phase solide (SPE) de colonne dans les canaux microfluidiques en plastique La fabrication de la phase solide est un processus en deux étapes. Au début, les microcanaux sont fabriqués à surface modifiée afin d&#39;améliorer l&#39;adhérence de la colonne SPE à l&#39;appareil en plastique. Greffage via photopolymérisation de surface est utilisée pour fonctionnaliser les murs des microcanaux polymériques. La procédure de greffage est comme suit: <ol><li> Les microcanaux sont remplis d&#39;un mélange 1:1 de diacrylate d&#39;éthylène (AED) et le méthacrylate de méthyle (MMA) avec de la benzophénone 3%, ce qui est un photo-initiateur d&#39;hydrogène abstraire. L&#39;AED, MMA et la benzophénone sont achetés chez Sigma-Aldrich, St. Louis, MO. </li><li> La puce est ensuite irradiée aux UV pendant 10 min à 254 nm de longueur d&#39;onde UV et 200 mJ / cm 2 d&#39;énergie dans un instrument exposition aux ultraviolets (CL-1000 Crosslinker UV, UPV Inc, Upland, CA). Le greffage se fait par H-abstraction de polymérisation et les résultats dans des chaînes de polymère en surface captif, qui se incorporé dans le mélange de polymérisation utilisé pour la préparation du monolithe dans la deuxième étape. </li><li> Le monomère en excès est éliminé par les voies d&#39;un rinçage avec 15 uL de méthanol en utilisant une aspiration. </li></ol> Après le processus de photografting, un polymère microporeux monolithe est formé dans les microcanaux qui piège les particules de silice pour l&#39;extraction de l&#39;ADN. Le monolithe poreux est préparé in situ comme suit: <ol><li> Les canaux à surface modifiée sont remplis d&#39;un mélange précurseur monolithe composé de BuMA (15% en poids), l&#39;EDMA (10% en poids), le 1-dodécanol (52,5% en poids), le cyclohexanol (22,5% en poids), DTSC (1% en poids par rapport aux monomères) et 0,7 um des particules de silice (15% par rapport au volume total du mélange de pré-polymère). Les microsphères de silice ont été achetés auprès de Polysciences, Inc (Warrington, PA) et les monomères et les solvants porogène sont achetés auprès de Sigma-Aldrich, St. Louis, MO. </li><li> La puce est irradié par UV dans le réticulant UV à 200 mJ / cm 2 pour les 1,1 min pour initier la polymérisation, et le monomère en excès est éliminé par les voies d&#39;un rinçage avec 15 uL de méthanol en utilisant une aspiration. </li><li> Les connexions fluidiques (NanoPorts connexions MC, Upchurch Scientific, Oak Harbor, WA) sont ensuite attachés à l&#39;introduction et les puits de collecte des canaux microfluidiques. </li><li> Le monolithe poreux est ensuite lavé à nouveau avec du méthanol pendant au moins 30 min en utilisant la pompe à seringue à un débit de 100mL/hour. </li></ol> La fabrication des CNT lyse monolithe dans les canaux microfluidiques en plastique Fabrication de la CNT de lyse (nanotubes de carbone) monolithe est un processus en deux étapes. Tout d&#39;abord, les microcanaux sont fabriqués à surface modifiée (greffé) de la même manière que pour les colonnes SPE dans le dispositif en plastique. Après le processus de photografting, un polymère microporeux monolithe est formé au sein du microcanal qui est imprégné de nanotubes de carbone multi-parois. Le monolithe poreux est préparé in situ comme suit: <ol><li> Nanotubes à parois multiples sont remis en suspension dans du cyclohexanol à une concentration de 2.27M. Cette remise en suspension est ultrasons pendant 30 minutes à une amplitude de 50% et 50% de cycle avec une cellule sonificateur perturbateur (Sonifier S-250D, fabriqué par ultrasons Branson, Danbury, CT). La sonication a fourni une suspension effective et stable 0.5M de NTC. Les NTC ont été achetés auprès Nanolabs (Brighton, MA). </li><li> Les canaux à surface modifiée sont remplies d&#39;un mélange précurseur de monolithe similaire, sauf que maintenant la solution du cyclohexanol + NTC est utilisé. Le mélange se compose de monolithe BuMA (15% en poids), l&#39;EDMA (10% en poids), le 1-dodécanol (52,5% en poids), et le cyclohexanol + NTC (22,5% en poids). Pour cette solution, le DTSC photoinitiateur est ajouté (1,13% en poids par rapport aux monomères). Les monomères, solvants porogène et DMPAPA sont achetés chez Sigma-Aldrich, St. Louis, MO. </li><li> La puce est irradié par UV dans le réticulant UV à 120 mJ / cm 2 pour les 0,9 min pour initier la polymérisation. L&#39;appareil est retourné à 180 degrés dans le crossliker et irradié pour un autre 0,9 min à 120 mJ / cm 2. Le monomère en excès est éliminé par les voies d&#39;un rinçage avec 50 ul de méthanol en utilisant une aspiration. </li><li> Les connexions fluidiques (NanoPorts connexions MC, Upchurch Scientific, Oak Harbor, WA) sont ensuite attachés à l&#39;introduction et les puits de collecte des canaux microfluidiques. </li></ol> Préparation des échantillons bactériens pour une utilisation avec la CNT lyse monolithe La préparation des échantillons pour un échantillon bactérien consiste à prendre une mesure de densité optique à 600 nm afin de s&#39;assurer que l&#39;échantillon n&#39;est pas très concentrée de manière à éviter l&#39;encrassement du canal. <ol><li> Prenez une lecture de DO à 600nm avec la bactérie dans les médias, cela se fait avec une BioPhotometer (Eppendorf BioPhotometer, Eppendorf Scientific, Inc, Westbury, NY). Diluer l&#39;échantillon jusqu&#39;à ce que la lecture OD tombent dans la fourchette de 0,18 à 0,25 A. </li><li> Centrifuger l&#39;échantillon à 6500 rpm pendant 5 minutes et décanter le surnageant. </li></ol> Lyse <ol><li> Remettre en suspension dans les bactéries GuSCN * + 0,01% SDS + 4% protéinase K (0.8mg/ml). Brièvement au vortex (1ml de bactéries dans les médias doivent être remises en suspension dans 1ml GuSCN). Protéinase K a été ajouté à la solution à l&#39;aide avec dénaturation des protéines qui sont attachées à l&#39;ADN bactérien et d&#39;inhiber DNAases et RNAases. Un avantage supplémentaire de la protéinase K est que cela aide aussi à briser les protéines de la paroi cellulaire qui est souhaitable car les deux bactéries gram-positives et gram-négatives murs bactéries cellules contiennent de grandes quantités de protéines (bien que les bactéries Gram négatif ont généralement plus de protéines ). Le tampon chaotrope aides outre, la protéinase K avec dénaturation des protéines tandis que les détergents perturbe la paroi cellulaire. Protéinase K et de thiocyanate de guanidinium (GuSCN) ont été achetés auprès de Qiagen Inc (Valencia, CA). SDS (dodécyl sulfate de sodium) a été acheté auprès de Pierce (Rockford, IL). </li><li> Immédiatement charge de l&#39;échantillon dans une seringue reliée à un tuyau en PEEK. Aspirer la seringue afin que l&#39;air n&#39;est pas dans le système. Branchez le tuyau à la nanoport et le canal. </li></ol> Une pompe à seringue KDS100 (fabriqué par KD Scientific, Holliston, MA) a été utilisée pour les expériences et le débit utilisé est de 450 ul / heure pour toutes les étapes. Débit du canal lyse de l&#39;échantillon au 450ul/hr. <ol><li> Recueillir l&#39;échantillon et de procéder à l&#39;extraction de l&#39;ADN par l&#39;intermédiaire de SPE (voir protocole d&#39;extraction d&#39;ADN). Remarque: Cet exemple bactérienne pas besoin de suivre l&#39;étape 2 de charge, depuis la suspension est GuSCN *. </li></ol> Protocole d&#39;extraction d&#39;ADN La procédure d&#39;extraction se compose de 3 étapes: <ol><li> Charger. </li><li> Wash </li><li> Éluer. </li></ol> Seringue KDS100 (fabriqué par KD Scientific, Holliston, MA) a été utilisée pour les expériences et le débit utilisé est de 300 ul / heure pour toutes les étapes. Charge <ol><li> Condition du canal avec le tampon chaotrope (GuSCN *, thiocyanate de guanidinium) pendant 1-2 min avant de commencer l&#39;expérience. </li><li> Mélanger l&#39;échantillon avec le tampon chaotrope dans le rapport 1:1. </li><li> Écoulement du mélange de l&#39;échantillon à travers le canal pendant 3 min. </li></ol> Laver <ol><li> Laver le canal avec de l&#39;éthanol 70% pendant 2 min. </li><li> Laver le canal avec de l&#39;éthanol à 100% pendant 2 min. </li></ol> Eluer <ol><li> Éluer l&#39;ADN extrait dans de l&#39;eau millipore pendant 3 min. Collectez 15 uL d&#39;purifiée, PCR-prêts ADN. </li></ol> * Le GuSCN du kit Qiagen (tampon RLT) a été utilisé.

<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

Dans notre laboratoire, nous avons réussi à isoler les acides nucléiques directement à partir de micro-volumes et submicroliter de sang, d&#39;urine et de selles humaines en utilisant un polymère / nanoparticules composites micro-lyse et solide colonnes d&#39;extraction en phase. Les échantillons récupérés sont concentrés, des échantillons de faible volume qui sont PCRable, sans aucun nettoyage supplémentaire. Ici, nous montrons comment fabriquer les puces microfluidiques thermoplastiques à l&#39;aide de gaufrage à chaud et l&#39;étanchéité de la chaleur. Ensuite, nous montrent comment utiliser la lumière à la surface in situ réalisé la greffe et la polymérisation à travers la puce scellée pour former des colonnes composites en phase solide. Nous démontrons le greffage et la polymérisation d&#39;un nanotube de carbone / polymère colonne composite pour la lyse des cellules bactériennes. Nous montrons ensuite le processus de lyse suivie par extraction en phase solide des acides nucléiques de l&#39;échantillon sur la puce en utilisant une silice / polymère colonne composite. Les protocoles joints contiennent des instructions détaillées sur la façon de faire à la fois la lyse et l&#39;extraction en phase solide colonnes.

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

Fabrication of the Thermoplastic Microfluidic Channels

Ici, nous montrer comment fabriquer des puces microfluidiques thermoplastiques en utilisant gaufrage à chaud et l&#39;étanchéité de la chaleur. Ensuite, nous montrent comment utiliser la lumière à la surface in situ réalisé la greffe et la polymérisation à travers la puce scellée pour former des colonnes composites en phase solide.

La fabrication des canaux microfluidiques thermoplastique

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

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JoVE Journal

Biology

13.3K vues.  Boston University. Ici, nous montrer comment fabriquer des puces microfluidiques thermoplastiques en utilisant gaufrage à chaud et l'étanchéité de la chaleur. Ensuite, nous montrent comment utiliser la lumière à la surface in situ réalisé la greffe et la polymérisation à travers la puce scellée pour former des colonnes composites en phase solide.

Vidéo: 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.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

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

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Biologie

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 du plasma Collage de PDMS pour fabrication bon marché de dispositifs microfluidiques

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.

Applications microfluidiques pour Diagnostics jetables

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.

Enregistrement Patch Clamp des canaux ioniques exprimés dans les oocytes de 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.

Fabrication d&#39;électrodes ampérométriques

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].

Fabrication de constructions myogéniques de l&#39;ingénierie tissulaire

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.

Protéomique pour identifier les protéines interagissant avec P2X2 canaux cationiques ligand-dépendants

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 sonde microfluidique: Fonctionnement et utilisation pour le traitement de surface localisé

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.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

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 et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

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