Ringraziamo il Dott. Aaron P. Fox presso l&#39;Università di Chicago, nel cui laboratorio di questo protocollo è stato originariamente progettato, e William Roden (Saint Louis University) per un ulteriore sviluppo del protocollo. Ringraziamo la Fondazione Whitehall e National Science Foundation per il supporto.

Parte 1: Preparazione fibra di carbonio <ol><li> Tagliare un fascio di fibre di carbonio (usiamo 7μm diametro) ad una lunghezza che è ~ 1,25 X la lunghezza della pipetta capillare. </li><li> Rimuovere il dimensionamento ~ facendo bollire 100 ml di acetone in un contenitore e aggiungendo le fibre al caldo, acetone bollente per 30 minuti o più. </li><li> Rimuovere fibre della acetone caldo, e trasferirli in un bicchiere pulito con 50-100 ml di acetone fresca. </li><li> Fibre a secco ponendo il fascio su un piatto di 150 millimetri aperto Petri e airdry durante la notte. </li></ol> Parte 2: Fuoco-lucidatura tubi di vetro capillare <ol><li> Fire-lucidare ogni tubo capillare (usiamo tubi capillari micro-ematocrito, 1,5 millimetri di diametro esterno, diametro interno 1,3 millimetri, 7,5 centimetri di lunghezza) tenendo il tubo in centro con una pinza e di esporre la fine della flame.Rotate il tubo stesso sul 3 volte, mentre nella fiamma. </li><li> Invertire il tubo e l&#39;estremità opposta nella fiamma, ancora con rotazione 3 volte. </li><li> Mettere al fuoco lucidato tubi in un piatto di vetro e trasferimento in forno fino a quando non sono pronti per essere riempito con fibre di carbonio. </li></ol> Parte 3: Threading una fibra di carbonio in un tubo capillare <ol><li> Simile alle descrizioni da Machado et al. (2008) e Mundroff e Wightman (2002), succhiare la fibra nel tubo di vetro con tubo di plastica collegato ad un vuoto con un filtro. </li><li> Tagliare la fibra con un scalpel.Leave ¼ di pollice in fibra di tubo. </li></ol> Parte 4: Tirare Pipettare-fibra in un elettrodo Puller <ol><li> Luogo tubo capillare nel estrattore elettrodo (usiamo Narishige Giappone Modello PP-830) e serrare le manopole di tenere il tubo capillare sul posto. </li><li> Manipolare gli interruttori per eseguire due tira, insieme con le impostazioni ottimali estrattore. </li><li> Una volta che la pipetta è tirato a metà, separare le due metà tagliando la fibra nuda al centro con le forbici. </li><li> Rimuovere ogni metà degli elettrodi e tirare la fibra sporgente per rompere il seal.Trim la fibra di vetro con le forbici in modo che solo 1 / 8 pollici di fibra è lasciato al di fuori del tubo. </li><li> Ripetere i passi 1-4 fino a circa 16 fibre elettrodi sono tirati. </li></ol> Parte 5: Esecuzione di resina epossidica-Glass Seal <ol><li> Scaldare una pre-made aliquota di resina epossidica su un piatto caldo insieme a 55 ° C per 6 minuti in una cappa aspirante. </li><li> Mentre la resina epossidica è il riscaldamento, avvolgere ~ 16 degli elettrodi tirato in un fascio utilizzando del nastro adesivo. </li><li> Dopo la resina Epon viene riscaldata per 6 minuti, aggiungere 0,7 grammi XOF MPDA alla resina calda Epon (14% w / v). Mescolare agitando il flacone. </li><li> Immergere il fascio di elettrodi in resina epossidica a caldo più solution.After indurente circa 20 secondi, una quantità sufficiente di resina epossidica entreranno le punte di vetro attraverso un&#39;azione capillare. </li><li> Annullare l&#39;, elettrodi posto pacco di cartone con su slot e posto questo vassoio in forno a 100 ° F. </li><li> Dopo almeno 48 ore, esaminare ogni elettrodo sotto un elettrodi microscope.Discard dissezione che non si riempiono di resina epossidica, hanno una punta fracassato, avere più di una fibra, etc.After ispezionando ogni elettrodo, posto di nuovo sul supporto di cartone e ritorno nel oven.Electrodes sono generalmente buoni per circa 1 mese dopo la fabbricazione. </li></ol> Parte 6: Taglio della fibra di carbonio <ol><li> In fibra di carbonio elettrodo posto attenzione su un vetrino con nastro di teflon. </li><li> Visualizza la punta sotto un microscopio da dissezione. </li><li> Utilizzare una lama pulita bisturi per tagliare perpendicolarmente attraverso la fibra di carbonio a lasciare una superficie piana pulita per il posizionamento della fibra di carbonio contro una cella. </li></ol>

<ol>
	<li>Mundorf, M. L., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amperometry+and+cyclic+voltammetry+with+carbon+fiber+microelectrodes+at+single+cells.">Amperometry and cyclic voltammetry with carbon fiber microelectrodes at single cells.</a> Curr Protocols in Neurosci. 6 (14), 1-6 (2002).</li><li>Machado, D. J., Montesinos, M. S., Borges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+practices+in+single-cell+amperometry.">Good practices in single-cell amperometry.</a> Methods in Molec. Biol. 440, 297-313 (2008).</li></ol>

Questo protocollo descrive come generare e tagliare elettrodi amperometrici. Durante le registrazioni amperometrica, un elettrodo di fibra di carbonio è posto contro la superficie di una cellula secretoria. Attività esocitotico si osserva come picchi amperometrico, che indica la corrente elettrochimica causata dal trasferimento di elettroni dopo l&#39;ossidazione delle catecolamine. Con la pratica facendo e taglio elettrodi amperometrici, i livelli di rumore può essere notevolmente migliorato rispetto a molti degli e...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Epoxy Hardener 1,3-phenylene-diamene flakes</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>P23954-100G04 716AJ</td>
<td>Aliquots are stored in a dessicator and should not be exposed to light.</td>
</tr>
<tr>
<td>Carbon Fibers</td>
<td>Material</td>
<td>Goodfellow</td>
<td>C005725</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Hematocrit Capillary Tubes</td>
<td>Material</td>
<td>Fisher</td>
<td>NC9836768</td>
<td>&nbsp;</td>
<tr>
<td>Epoxy Resin</td>
<td>Reagent</td>
<td>Miller-Stephenson</td>
<td>Epon Resin 828</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Electrode Puller</td>
<td>Instrument</td>
<td>Narishige Japan</td>
<td>PP-830</td>
<td>1st pull = 24.8 degrees C; 2nd pull = 62.7 degrees C</td>
</tr>
<tr>
<td>Oven</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set to 100 degrees F.</td>
</tr>
<tr>
<td>Hot plate</td>
<td>Instrument</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>set to 55 degrees C</td>
</tr>		</tbody>
		</table>
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fabrication of amperometric electrodes

Elettrodi in fibra di carbonio sono cruciali per la determinazione del rilascio di catecolamine dalle vescicole in singole cellule per le misure amperometria. Qui, descriviamo le tecniche necessarie per generare rumore basso (&lt;0,5 pA) elettrodi. Le tecniche sono stati modificati dalle descrizioni pubblicato dai ricercatori precedenti (1,2). Gli elettrodi sono fatti con la preparazione delle fibre di carbonio e filettatura individualmente in ciascun tubo capillare utilizzando un vuoto con un filtro per aspirare la fibra. Successivamente, il tubo capillare con la fibra è tirato da un elettrodo estrattore, la creazione di due metà, ognuna con una punta sottile. Gli elettrodi sono immersi in acqua calda, epossidico liquido mescolato con catalizzatore per creare un sigillo di vetro-resina epossidica. Infine, gli elettrodi sono posti in un forno per curare la resina epossidica. Attenta gestione degli elettrodi è fondamentale per garantire che siano fatte in modo coerente e senza danni. Questo protocollo mostra come fabbricare e tagliare elettrodi amperometrici per la registrazione da singole cellule.

Watch this Scientific Journal Video about Fabrication of Amperometric Electrodes at JoVE.com

Fabrication of Amperometric Electrodes

Questo protocollo descrive come generare elettrodi in fibra di carbonio. Gli elettrodi vengono successivamente utilizzate per rilevare rilascio di catecolamine dalle vescicole con amperometria in fibra di carbonio.

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 ...

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Electrochemical Analyses and Redox Titration

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14.3K Views.  Saint Louis University School of Medicine. Questo protocollo descrive come generare elettrodi in fibra di carbonio. Gli elettrodi vengono successivamente utilizzate per rilevare rilascio di catecolamine dalle vescicole con amperometria in fibra di carbonio.

Video: Fabrication of Amperometric Electrodes

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Applicazione della Luce cured resina adesiva dentale per montaggio elettrodi o sonde Microdialisi negli esperimenti cronica

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Ricerca

Biologia

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Cellule patterning su elettrodi Tin otticamente trasparente di ossido di indio

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

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 ...

PDMS Device Fabrication and Surface Modification

PDMS fabbricazione del dispositivo e la modifica della superficie

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 ...

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

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 ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Rendendo Patch-pipette e Elettrodi Sharp con un estrattore programmabile

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Micro-drive array per la cronica In vivo</emRegistrazione>: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

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 ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

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

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

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

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

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