Die mikrofluidischen Kanälen sind in einem zyklischen Polyolefin (Zeonex ® 690R, Zeon Chemical Inc., Louisville, KY) hergestellt. Zeonex hat eine Glasübergangstemperatur (T g) von 136 ° C und ist UV-durchlässig, das ist wichtig für das Licht gerichtet Chemie in die poröse Monolith Bildung verwendet. Die Mikrokanäle werden durch Mikro-Heißprägen mit einem NiCo elektrogeformte Master-Form, die die negative (inverse) Merkmale des mikrofluidischen Plattform hergestellt. Das Heißprägen wird wie folgt durchgeführt: <ol><li> Die Form und das Polymer Substrat zwischen zwei parallelen Platten aus Metall angebracht. </li><li> Die Platten sind bis zum Prägetemperatur (166 ° C), die 30 º C über dem T g Zeonex erhitzt wird. </li><li> Die Platten werden dann zusammen für ca. 5 min gepresst und der Druck wird bei 250 psi gehalten. </li><li> Die Form und das Substrat werden dann von der beheizten Walzen, dürfen für 1-2 min abkühlen entfernt, und dann manuell voneinander getrennt. </li><li> Wells von 1,5-mm-Durchmesser an den Enden der Kanäle für die Probenvorbereitung Einführung und Erhebung gebohrt. </li><li> Die geprägten Kunststoff-Substrat wird dann thermisch mit einem anderen Stück Zeonex verklebt geschlossenen mikrofluidischen Kanälen bilden. Die geprägten Substrat und die Abdeckung Stück kann UV-Ozon-oder Plasma vor der thermischen Anbindung an die Abdichtung Effizienz zu steigern und sicherzustellen, dass die Treue der beiliegenden Kanalstruktur (Bhattacharyya und Klapperich, 2007) behandelt werden. </li></ol> Herstellung von Festphasenextraktion (SPE) Spalte innerhalb der Kunststoff-mikrofluidischen Kanälen Die Herstellung der festen Phase ist ein zweistufiger Prozess. Zunächst werden die gefertigten Mikrokanäle Oberfläche modifiziert werden, um die Haftung der SPE-Säule auf der Kunststoff-Vorrichtung zu verbessern. Grafting via Oberfläche Photopolymerisation wird verwendet, um die Wände des polymeren Mikrokanäle funktionalisieren. Die Pfropfung ist wie folgt: <ol><li> Die Mikrokanäle sind mit einem 1:1-Gemisch aus Ethylendiacrylat (EDA) und Methylmethacrylat (MMA) mit 3% Benzophenon, das ein Wasserstoffatom abstrahieren Photoinitiator gefüllt ist. Das EDA, MMA und Benzophenon sind von Sigma-Aldrich, St. Louis, MO gekauft. </li><li> Der Chip wird dann UV-Bestrahlung für 10 min bei 254 nm UV-Wellenlänge und 200 mJ / cm 2 Energie in eine UV-Exposition Instrument (CL-1000 UV Crosslinker, UPV Inc., Upland, CA). Die Pfropfung erfolgt über H-Abstraktion Polymerisation und die Ergebnisse in der Oberfläche angebundenen Polymerketten, die in der Polymerisationsmischung für die Vorbereitung des Monolithen in der zweiten Stufe verwendet einzubringen. </li><li> Das überschüssige Monomer wird aus den Kanälen durch Spülen mit 15 ul Methanol unter Vakuum abgesaugt. </li></ol> Nach dem photografting Verfahren wird eine mikroporöse Polymer-Monolith innerhalb der Mikrokanäle gebildet, fangen die Silica-Teilchen zur DNA-Extraktion. Die poröse Monolith ist in situ hergestellt wie folgt: <ol><li> Die Oberfläche verändert Kanäle sind mit einem Monolith Precursormischung bestehend aus Buma (15% wt), EDMA (10% wt), 1-Dodecanol (52,5% wt), Cyclohexanol (22,5% Gew.), DMPAP (1 Gew.% in Bezug gefüllt Monomere) und 0,7 um Silica-Partikel (15% bezogen auf das Gesamtvolumen der Pre-Polymer-Mischung). Die Silica-Mikrokugeln wurden von Polysciences, Inc. (Warrington, PA) gekauft und die Monomere und porogenen Lösungsmittel sind von Sigma-Aldrich, St. Louis, MO gekauft. </li><li> Der Chip wird mit UV im UV-Crosslinker bei 200 mJ / cm 2 für 1,1 min, um die Polymerisation zu initiieren bestrahlt, und das überschüssige Monomer wird aus den Kanälen durch Spülen mit 15 ul Methanol unter Vakuum abgesaugt. </li><li> Die fluidischen Verbindungen (NanoPorts TM-Verbindungen, Upchurch Scientific, Oak Harbor, WA) werden dann über die Einführung und Erhebung Vertiefungen der mikrofluidischen Kanälen verbunden. </li><li> Die poröse Monolith wird dann wieder mit Methanol für mindestens 30 min mit der Spritzenpumpe bei einer Flussrate von 100mL/hour gewaschen. </li></ol> Herstellung von CNT Lyse Monolith in der Kunststoff-mikrofluidischen Kanälen Die Herstellung der CNT (Kohlenstoff-Nanoröhrchen) Lyse Monolith ist ein zweistufiger Prozess. Zunächst werden die gefertigten Mikrokanäle Oberfläche modifiziert (gepfropft) in der gleichen Weise wie für die SPE-Säulen in der Kunststoff-Gerät. Nach dem photografting Verfahren wird eine mikroporöse Polymer-Monolith innerhalb des Mikrokanals, die mit Multi-Walled Carbon Nanotubes imprägniert gebildet wird. Die poröse Monolith ist in situ hergestellt wie folgt: <ol><li> Multi-Walled-Nanotubes sind in Cyclohexanol in einer Konzentration von 2,27 resuspendiert. Diese Resuspension wird für 30 Minuten bei 50% Amplitude Ultraschall und 50% ED mit einem sonifier Cell Disruptor (Sonifier S-250D, von Branson Ultraschall, Danbury, CT) hergestellt. Die Beschallung ein wirksames und stabiles 0.5M Suspension von CNTs. Die CNTs wurden aus Nanolabs (Brighton, MA) erworben. </li><li> Die Oberfläche verändert Kanäle sind mit einer ähnlichen Monolithen Precursormischung außer dass jetzt die Lösung von Cyclohexanol + CNTs wird gefüllt. Der Monolith Mischung besteht aus Buma (15% wt), EDMA (10% wt), 1-Dodecanol (52,5% Gew.) und Cyclohexanol + CNTs (22,5% wt). Zu dieser Lösung der Photoinitiator DMPAP hinzugefügt wird (1,13% Gew. mit Bezug auf Monomere). Die Monomere, porogenen Lösungsmitteln und DMPAPA sind von Sigma-Aldrich, St. Louis, MO gekauft. </li><li> Der Chip wird mit UV im UV-Crosslinker bei 120 mJ / cm 2 für 0,9 min, um die Polymerisation zu initiieren bestrahlt. Das Gerät ist um 180 Grad gedreht in der crossliker und bestrahlt weitere 0,9 min bei 120 mJ / cm 2. Das überschüssige Monomer wird aus den Kanälen durch Spülen mit 50 ul Methanol unter Vakuum abgesaugt. </li><li> Die fluidischen Verbindungen (NanoPorts TM-Verbindungen, Upchurch Scientific, Oak Harbor, WA) werden dann über die Einführung und Erhebung Vertiefungen der mikrofluidischen Kanälen verbunden. </li></ol> Bakterielle Probenvorbereitung für die Verwendung mit CNT Lyse Monolith Die Probenvorbereitung für eine bakterielle Probe die Einnahme einer optischen Dichte-Messung bei 600nm, um sicherzustellen, dass die Probe nicht extrem, um zu verhindern Verstopfung des Kanals konzentriert. <ol><li> Nehmen Sie eine OD Lesen bei 600nm mit den Bakterien in den Medien, ist dies mit einem BioPhotometer (Eppendorf BioPhotometer, Eppendorf Scientific, Inc., Westbury, NY) durchgeführt. Verdünnen Sie die Probe, bis die OD im Bereich 0,18-0,25 A. Herbst </li><li> Zentrifugieren Sie die Probe bei 6.500 rpm für 5 Minuten und Dekantieren des Überstandes. </li></ol> Lysis <ol><li> Resuspendieren Bakterien in GuSCN * + .01% SDS + 4% Proteinase K (0.8mg/ml). Vortex kurz (1 ml der Bakterien in Medien sollten in 1ml GuSCN resuspendiert werden). Proteinase K wurde zu der Lösung gegeben, um mit Denaturierung von Proteinen, die an der bakteriellen DNA gebunden sind und DNasen und RNAasen verhindern helfen. Ein weiterer Vorteil der Proteinase K ist, dass es hilft auch mit Abbau der Zellwand Proteine, die wünschenswert ist, da sowohl gram-positive Bakterien und gram-negative Bakterien Zellwände große Mengen an Protein (enthält zwar gram-negative Bakterien in der Regel mehr Eiweiß haben ). Die chaotropen Puffer zusätzlich unterstützt die Proteinase K mit Denaturierung von Proteinen während des Waschmittels stört die Zellwand. Proteinase K und Guanidiniumthiocyanat (GuSCN) wurden von Qiagen Inc. (Valencia, CA) gekauft. SDS (Natriumdodecylsulfat) wurde von Pierce (Rockford, IL) erworben. </li><li> Unmittelbar Belastung der Probe in einer Spritze verbunden PEEK-Schlauch. Saugen Sie die Spritze so, dass keine Luft in das System. Verbinden Sie den Schlauch an den nanoport und Kanal. </li></ol> Ein KDS100 Spritzenpumpe (hergestellt von KD Scientific, Holliston, MA) wurde für die Experimente verwendet und die Durchflussmenge eingesetzt ist 450 ul / Stunde für alle Schritte. Durchfluss der Probe Lyse-Kanal bei 450ul/hr. <ol><li> Sammeln Sie die Probe und gehen Sie auf die DNA-Extraktion mittels SPE (siehe DNA-Extraktion-Protokoll). HINWEIS: Diese bakterielle Probe müssen nicht laden Schritt 2 folgen, da die Suspension GuSCN *. </li></ol> DNA-Extraktion-Protokoll Das Extraktionsverfahren besteht aus 3 Schritten: <ol><li> Load. </li><li> Washington </li><li> Eluieren. </li></ol> KDS100 Spritzenpumpe (hergestellt von KD Scientific, Holliston, MA) wurde für die Experimente verwendet und die Durchflussmenge eingesetzt wurde 300 ul / Stunde für alle Schritte. Last <ol><li> Zustand des Kanals mit chaotropen Puffer (GuSCN *, Guanidiniumthiocyanat) für 1-2 min vor dem Start des Experiments. </li><li> Mischen Sie die Probe mit chaotropen Puffer im Verhältnis 1:1. </li><li> Flow-Probe-Gemisch durch den Kanal für 3 min. </li></ol> Waschen <ol><li> Waschen Sie den Kanal mit 70% Ethanol für 2 min. </li><li> Waschen Sie den Kanal mit 100% Ethanol für 2 min. </li></ol> Eluieren <ol><li> Eluieren der extrahierten DNA in Millipore-Wasser für 3 min. Sammeln Sie 15 ul des gereinigten PCR-ready DNA. </li></ol> * Die GuSCN aus dem Qiagen-Kit (Buffer RLT) verwendet wurde.

<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

In unserem Labor haben wir erfolgreich Nukleinsäuren direkt von Mikroliter-und Submikroliter Mengen von menschlichem Blut, Urin und Stuhl unter Verwendung von Polymer / Nanopartikel-Composite mikroskaligen Lyse und Festphasenextraktion Säulen isoliert. Die gewonnene Proben konzentriert, kleinen Probenvolumina, dass PCRable werden, ohne zusätzliche Aufräumarbeiten. Hier zeigen wir, wie thermoplastische Mikrofluidik-Chips mit Heißprägen und Wärmeversiegelung herzustellen. Dann zeigen wir, wie man in situ Licht gerichtet Oberflächenpfropfung und Polymerisation durch die versiegelten Chip verwenden, um den Verbund Festphase Spalten bilden. Wir zeigen, Pfropfen und Polymerisation von einem Kohlenstoff-Nanoröhrchen / Polymer-Verbundstoff Spalte für die bakterielle Zell-Lyse. Wir zeigen dann die Lyse-Prozess durch Festphasen Extraktion von Nukleinsäuren aus der Probe auf dem Chip mit einem Silica / Polymer-Verbundstoff-Säule. Die beigefügte Protokolle enthalten detaillierte Anweisungen, wie Sie beide Lyse und Festphasenextraktion Spalten zu machen.

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

Fabrication of the Thermoplastic Microfluidic Channels

Hier zeigen wir, wie thermoplastische Mikrofluidik-Chips mit Heißprägen und Wärmeversiegelung herzustellen. Dann werden wir zeigen, wie man in situ Licht gerichtet Oberflächenpfropfung und Polymerisation durch die versiegelten Chip verwenden, um den Verbund Festphase Spalten bilden.

Die Herstellung der thermoplastischen Mikrofluidikkanälen

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

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K Aufrufe.  Boston University. Hier zeigen wir, wie thermoplastische Mikrofluidik-Chips mit Heißprägen und Wärmeversiegelung herzustellen. Dann werden wir zeigen, wie man in situ Licht gerichtet Oberflächenpfropfung und Polymerisation durch die versiegelten Chip verwenden, um den Verbund Festphase Spalten bilden.

Serie: 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.

Herstellung eines mikrofluidischen Vorrichtung für die Abschottung der Neuron Soma und Axonen

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

Forschung

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-Plasma-Bonding von PDMS für preiswerte Herstellung von Mikrofluidik-Geräte

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.

Mikrofluidik-Anwendungen für Einweg-Diagnostics

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 Clamp Recording von Ionenkanälen in Xenopus Oozyten exprimiert

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.

Herstellung von amperometrische Elektroden

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

Fabrication of Myogenic Engineered Tissue Constructs

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

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

Herstellung von Myogenic Engineered Tissue Konstrukte

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

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.

Proteomics, um Proteine ​​interagieren mit P2X2 Ligand-Gated Kationenkanäle Identifizieren

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.

Die Mikrofluidik-Probe: Betrieb und Nutzung für lokalisierte Oberflächenbearbeitung

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

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.

Mutagenese und funktionale Analyse von Ionenkanälen heterolog in Säugerzellen exprimierten

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-und Perfusion Techniken zur Untersuchung von Ionenkanälen ausgedrückt in Xenopus Oozyten

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