La pressione di lucidatura tecnica è stata sviluppata in collaborazione con Lockery SR.

Pipette tirando Utilizzando un filamento estrattore come un Sutter P-97 Flaming Puller microelettrodo / Brown, tirare un insieme di circa 10-20 pipette. <ol><li> Seleziona il tuo bicchiere. Noi usiamo borosilicato (BF150-86-10, OD = 1,5 mm, ID = 0,86 mm). Conservare con cura il vetro in modo che rimane pulito e senza polvere. </li><li> Progettare un programma di trazione. L&#39;obiettivo primario è una punta di apertura circa la dimensione desiderata, che dipende dalla applicazione. Sutter P-97 Cookbook Pipettare 2 è un eccellente punto di riferimento. </li><li> Osservate la vostra pipette al microscopio per determinare diametro di apertura e morbidezza. Eliminare ruvido, consigli irregolare, o irregolari. Per la registrazione standard di patch clamp, un&#39;apertura punta che è di 2-4 micron tutto è meglio. Una punta smussata porta ad una minore resistenza per il diametro dell&#39;apertura stessa, che si preferisce. Forma e dimensioni possono essere modificate durante la lucidatura, di seguito. </li></ol> Pressione lucidatura pipette <ol><li> Collegare un titolare pipetta con una linea pulita ed aria compressa. Utilizzando un regolatore, impostare la pressione dell&#39;aria compressa linea a ~ 40 PSI. Collegare la linea di aria compressa ad una valvola a 4 vie di controllo che permetterà la pressione titolare pipetta ad essere attivata e disattivata. Collegare un titolare pipetta alla valvola di controllo con una lunghezza di ⅛ in Tygon ® tubazione. </li><li> Utilizzare un impianto di lucidatura (microforge) con un alta pressione titolare pipetta, una bobina di lucidatura controllato da un pedale, un microscopio equipaggiato con un ingrandimento basso e alto, lunga distanza (idealmente, 10x e 100x) aria obiettivi. E &#39;fondamentale essere in grado di osservare l&#39;apertura pipetta durante il protocollo. </li><li> Opzionale: Cappotto la pipetta con un isolante idrofoba per diminuire capacità e migliorare le caratteristiche del rumore (vedi &quot;pipette Pulling&quot; per questa tecnica). </li><li> Verificare che la valvola è in posizione off. Inserire una pipetta nel supporto pipetta e stringere il raccordo di a mano. Nota di sicurezza: se la pipetta venga mantenuta ferma nel supporto pipetta, la pressione dell&#39;aria provoca la pipetta di essere espulso dal titolare pipetta. </li><li> Posizionare la pipetta e titolare nel microforge. Posizionare il titolare pipetta sul palco microscopio in modo che sia la punta e la bobina lucidatura sono visibili in basso (10x) accendere il microforge. Portare la punta ~ 75 micron dal filamento. Tieni presente che il filamento si espande quando riscaldato. </li><li> Passare alla elevata potenza (100x) obiettivo. Sia la punta della pipetta e la bobina lucidatura dovrebbe essere a fuoco e nel campo di vista. In caso contrario, regolare l&#39;altezza della bobina lucidatura. (La posizione della pipetta non può essere regolata). La punta della pipetta e la bobina di lucidatura dovrebbe essere vicino ai lati opposti del campo visivo. Evitare di posizionare la bobina troppo vicino alla punta della pipetta, perché si espanderà in caso di riscaldamento e di rilievo nella vostra pipetta. </li><li> Girate la valvola per dirigere l&#39;aria pressurizzata nella pipetta, e il calore la punta della pipetta fino a quando il cono dietro la punta comincia a palloncino verso l&#39;esterno. </li><li> Interrompere il riscaldamento prima che la punta palloncini troppo ampiamente. La sperimentazione è necessaria per determinare la distanza appropriata, il calore, e il tempo per la forma desiderata. </li><li> Opzionale: ridurre il diametro di apertura punta da lucidare la punta, in assenza di pressione. I punti 7 e 8 può ripetere per mettere a punto sia la forma e le dimensioni della punta della pipetta. </li><li> Spegnere la pressione e puntando la punta della pipetta in una direzione sicura, rimuovere la pipetta e metterla in un contenitore pulito, dove sarà protetta dalla polvere. Nota di sicurezza: Se la pressione dell&#39;aria non è spento, la pipetta può essere espulso dal titolare pipetta durante la rimozione. </li></ol> Note sui microforge Usiamo un microforge assemblato da off-the-shelf parti disponibili in commercio. (È possibile anche acquistare un kit di lucidatura pressione da alcune aziende, ad esempio, ALA Scienza, GlassWorx). Indipendentemente dal microforge, è essenziale avere ottiche che sono buone abbastanza per vedere l&#39;apertura punta, che è spesso solo un paio di micron di diametro. Usiamo un lungo lavoro obiettivo 100x a distanza per questo scopo. Inoltre, il cappotto la bobina di lucidatura ad alta temperatura di fusione del vetro in modo da minimizzare il deposito di metallo vaporizzato sul pipette. Parte 3: risultati attesi Lucidatura moderata pressione può ridurre la resistenza di una pipetta senza modificare sensibilmente l&#39;apertura diametro. Se lo si desidera, questo protocollo può essere usato per fare pipette con sub-micron aperture e resistenze tra 5-10MΩ.

<ol>
	<li>Goodman, M. B., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+polishing:+a+method+for+re-shaping+patch+pipettes+during+fire+polishing. .">Pressure polishing: a method for re-shaping patch pipettes during fire polishing. .</a> J Neurosci Methods. 100, 13-15 (2000).</li></ol>

Inizialmente abbiamo sviluppato questo metodo per pipette utile per patch-clamp registrazione 2-3 micron neuroni in C. elegans 1. E &#39;in uso di routine nel laboratorio Goodman. Noi pipette pressione smalto non solo per la registrazione da piccolo C. elegans neuroni, ma anche per l&#39;ottenimento di registrazioni macropatch e single-channel dei canali ionici espressi in ovociti di Xenopus. Per un dato in punta di apertura del diametro, la forma smussata produce una minore resistenza pipetta. Resistenza pipett...

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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>
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<td>Glass Capillaries</td>
<td>Consumable</td>
<td>Sutter Instruments</td>
<td>BF150-85-10 (or similar)</td>
<td>The method works with any type of capillary except those made from quartz. However, we recommend using capillaries with a filament.</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>Microscope</td>
<td>Leica</td>
<td>DM IL (or similar)</td>
<td>Any simple inverted microscope will do. However, it is essential to have sufficient magnification to see pipettes during polishing. We use a 100x metallurgical lens with a long working distance for this purpose.</td>
</tr>
<tr>
<td>Microforge Kit</td>
<td>Tool</td>
<td>ALA Science</td>
<td>CPM-2</td>
<td>Supplies polishing coil, controller and foot pedal.</td>
</tr>
<tr>
<td>Air pressure source</td>
<td>Tool</td>
<td>ALA Science</td>
<td>PR-60</td>
<td>Accessory for providing high pressure, includes a suitable valve. Alternatively, compressed air from the building can be used together with a filter/regulator (set to 40 PSI) and a 4-way valve (see below)</td>
</tr>
<tr>
<td>4-way valve</td>
<td>Tool</td>
<td>Cole-Parmer</td>
<td>A-98150-01</td>
<td>Fittings will be needed to couple valve to tubing</td>
</tr>
<tr>
<td>Pipette Holder with Luer Connector</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH-6S20</td>
<td>Use a male luer-to-barb fitting to connect pipette holder to high-pressure tubing</td>
</tr>
<tr>
<td>Tygon&#174; tubing</td>
<td>Tool</td>
<td>Cole-Parmer</td>
<td>06408-62</td>
<td>Use this tubing to connect pipette holder to 4-way valve
</td>
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pressure-polishing pipettes for improved patch-clamp recording

Pressione lucidatura è un metodo per modellare pipette di vetro per patch-clamp registrazione. In primo luogo abbiamo sviluppato questo metodo per la realizzazione di pipette adatto per la registrazione di piccole dimensioni (&lt;3 m) corpi cellulari neuronali. Il principio di base è simile al vetro soffiato e combina la pressione dell&#39;aria e del calore per modificare la forma di pipette di patch preparata da un estrattore micropipetta convenzionale. Può essere applicato ai cosiddetti soft (calce sodata) e difficile (borosilicato) occhiali. In generale, la lucidatura della pressione può ridurre la resistenza pipetta del 25% senza diminuire il diametro dell&#39;apertura punta (Goodman e Lockery, 2000). Può essere applicato a qualsiasi tipo di vetro e richiede solo l&#39;aggiunta di un valvola di alta pressione e montato su un microforge. Questa tecnica è essenziale per la registrazione da cellule ultrasmall (&lt;5 m) e può anche migliorare singolo canale di registrazione, riducendo al minimo la resistenza pipetta. La forma smussata è utile anche per la registrazione perforata-patch clamp, perché i risultati in forma di questa punta una bozza membrana più disponibile per la perforazione.

Watch this Scientific Journal Video about Pressure-polishing Pipettes for Improved Patch-clamp Recording at JoVE.com

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Questa è una guida per modificare la forma di micropipette di vetro. In particolare, utilizzando il calore e la pressione dell&#39;aria la conicità è allargato senza aumentare l&#39;apertura punta, con conseguente minore resistenza pipetta. Questo è fondamentale per ottenere registrazioni a basso rumore di piccole cellule, ma è utile in molte applicazioni.

Pressione lucidatura Pipette per una migliore patch-clamp di registrazione

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

pressure-polishing-pipettes-for-improved-patch-clamp-recording

Research

JoVE Journal

Biology

12.9K Views.  Stanford University School of Medicine. Questa è una guida per modificare la forma di micropipette di vetro. In particolare, utilizzando il calore e la pressione dell'aria la conicità è allargato senza aumentare l'apertura punta, con conseguente minore resistenza pipetta. Questo è fondamentale per ottenere registrazioni a basso rumore di piccole cellule, ma è utile in molte applicazioni.

Video: Pressure-polishing Pipettes for Improved Patch-clamp Recording

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Metodi per la registrazioni Morsetto capacità patch dal Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Ricerca

Biologia

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

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

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

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

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

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 ...

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

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

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

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

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

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

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ubiquity of microbes creates a copious supply of potential contaminants in a laboratory. To ensure experimental success, the number of contaminants on equipment and work surfaces must be minimized. Common among many experiments in microbiology are techniques involving the measurement and transfer of cultures containing bacterial cells or viral particles. To do so without contacting non-sterile surfaces or contaminating sterile media requires (1) preparing a sterile workspace, (2) precisely setting and accurately reading instruments for aseptic transfer of liquids, and (3) properly manipulating instruments, cultures flasks, bottles and tubes within a sterile field. Learning these procedures calls for training and practice. At first, actions should be slow, deliberate, and controlled with the goal being for aseptic technique to become second nature when working at the bench. Here we present the steps for measuring volumes using serological pipettes and micropipettors within a sterile field created by a Bunsen burner. Volumes range from microliters (&mu;l) to milliliters (ml) depending on the instrument used. Liquids commonly transferred include sterile broth or chemical solutions as well as bacterial cultures and phage stocks. By following these procedures, students should be able to: &#8226;Work within the sterile field created by the Bunsen burner flame. &#8226;Use serological pipettes without compromising instrument sterility.&#8226; Aspirate liquids with serological pipettes, precisely reading calibrated volumes by aligning the meniscus formed by the liquid to the graduation marks on the pipette. &#8226;Keep culture bottles, flasks, tubes and their respective caps sterile during liquid transfers. &#8226;Identify different applications for plastic versus glass serological pipettes. &#8226;State accuracy limitations for micropipettors. &#8226;Precisely and accurately set volumes on micropipettors. &#8226;Know how to properly use the first and second stop on a micropipettor to aspirate and transfer correct volumes.

When working in a laboratory, it is imperative to minimize sources of contamination. Aseptic technique refers to procedures that permit transfer of cultures and reagents while avoiding contact with non-sterile surfaces. Serological pipettes and micropipettors are used to measure precise volumes without compromising sterility of solutions used in experiments.

Tecniche asettiche di laboratorio: Trasferimenti Volume con pipette sierologiche e Micropipettatrici

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Esplorare arteriosa Muscolo liscio KV7 Funzione canale del potassio con morsetto Elettrofisiologia Patch e Myography pressione

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Giant Preparazione liposomi per Immagini e di patch-clamp elettrofisiologia

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canale Cell-allegato patch-clamp registrazione

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Ricostituzione di una proteina transmembrana, la tensione gated canali ionici, KvAP, in Giant unilamellari vescicole per la microscopia e Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Implementazione Patch Clamp e vivo microscopia a fluorescenza per il monitoraggio delle proprietà funzionali di recente isolato PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...