Patch-Clamp-Setup: Optik: Die Optik ist sehr wichtig, eine gute Zelle zu finden, und um die Position der es zu etablieren. Versuchen Sie, ein klares Bild von den Zellen zu erhalten: scharfe Kanten, weich aussehende Zelle. Scan das gesamte Feld, für gute MNTB wichtigsten Zellen mit angeschlossenem Kelch zu suchen. Drehen Gericht für Kelche aus verschiedenen Blickwinkeln betrachten. Zunächst ist es OK, um auf Ballon-förmigen Klemme Praxis. Pick-Zellen an der Oberfläche des Slice, oder in der 2. Zellschicht. Ziel der ersten 1 / 3 rd der Zelle mit Pipette Identifizieren Hopfenzapfen: für ein Doppelzimmer Membran (siehe Abbildung 1) Look, und drehen Sie die Scheibe Gericht, es aus verschiedenen Blickwinkeln sehen, um zu bestätigen, dass es ein Kelch ist. <img src="/files/ftp_upload/old/244_2008_8_22_2/244_Figure-01.png" alt="Abbildung 1" width="298" height="159" /> Abbildung 1 Tiefe der Kelch: Calyx sollte auf der Oberfläche oder als nahe an der Oberfläche wie möglich. Zur Verfügung stehende Fläche für das Patchen: Verwenden Sie Fein-Fokus-Knopf am Mikroskop zu bestimmen, wie &quot;breit / tief&quot; der Kelch ist. Zum Beispiel werden ca. 10% einer vollen Umdrehung der Feinfokussierung Ihnen mehr als genug Fläche, um auf Patch. Abdichtung auf einen präsynaptischen Zelle: Um zu Zelle angebracht zu bekommen, für einen &quot;kleinen&quot; Verformung auf der Zelloberfläche durch pos Druck (~ 0.15psi) in die Pipette suchen, verwenden Sie Manipulator Pipette laufen nach oben, unten und quer, und sogar in der Oberfläche des presyn Terminal, um eine gute Verformung zu bekommen, dann lassen Sie Druck und Unterdruck anwenden, wenn und soweit notwendig. PULSE-, Datenerfassungs-Programm: Brauchen Sie, um Puffergröße vor dem ersten Gebrauch zu erhöhen, und wenn durch Stichproben Kapazität erforderlich. Brauchen Sie laden modifizierten Makros jeder Zeit. Flüssige Junction Potential: Presyn Recording Solution-11mV (negative 11mV). Presyn: Cslow = ~ 15 pF Rs comp für presyn: 10 us, 65% (mehr Entschädigung kann möglich w / out Schwingungen) Vor der Aufnahme Kapazität: Prüfstrom Entspannung (10 mV, 10 ms Sprung) zu sehen, ob Entspannung monoexponentielle ist, dh ob presyn Begriff, der von einem einzigen Innenraum modelliert werden kann. Filter: Set mit 30 kHz und 10 kHz (Bessel-Filter 1 und 2) während der Entspannung Mess-, um sicherzustellen, dass eine schnelle Komponente nicht auf verfehlt. Während Mini-Aufnahmen verwendet Xinsheng 10 kHz und 5 kHz. LGW hat mich gebeten, nur Filter 1 Satz bei 30 kHz verwenden, um sicherzustellen, dass wir Anstiegszeit von Mini messen. Dateien benennen: 7-stellig 000mmxx, wobei mm Monat (oder Monat und Jahr) und xx ist die Zellzahl. Cleaning Solution Lines: Zeilen am Ende des Experiments mit 20 ml 0,1 M HCl gereinigt, gefolgt von 200 ml ddH2O. Externe Lösungen recycelt werden, ist Pumpendrehzahl 20-30 Umdrehungen pro Minute. (Lösung der Rückkehr aus Bad Kammer fließt zurück in den Messzylinder). Set up 3 abgehende Leitungen, um eine ausreichende Saugleistung zu gewährleisten. Pipette Präsynaptische Recording Solution: ~ 310 Millisekunden osmolaren Presyn Lösung muss ~ 310 mOsm zu R-Zugang Anstieg im Experiment zu verhindern. Presyn Pipettenlösung unterscheidet sich von postsyn Pipettenlösung (Presyn hat ATP &amp; GTP für ex.). TTX-TEA-Lösung (Ca Strömen Isolat): <ul><li> Versuchen Sie, nach dem Versiegeln oder sogar Patches gelten, auf die Zelle, wie TEA in Lösung Blöcke K-Kanäle und depolarisiert die Zelle. Das ist nicht gut für die Zelle. Aus dem gleichen Grund ist es vielleicht nicht möglich sein, die Scheibe Wiederverwendung nach TTX angewendet wird. </li><li> Vergleichen Sie die erste und die letzte Antwort der Zelle, oder ein Stück für eine Wirkung anhaltender Depolarisation zu überprüfen. </li><li> TTX ist bei 1 uM statt 0,5 uM verwendet, so ist der Effekt schneller. Benötigen Sie weniger Zeit nach dem Wechsel Lösungen warten. </li></ul> Cutting Slices Vibratom Einstellungen: <ul><li> 70Hz seitlichen Schwingungen </li><li> Amplitude: 1,0 mm </li><li> Fahrgeschwindigkeit 0,01-0,02 mm / sec beim Schneiden Scheiben mit Kelch, 0,1 ansonsten Dicke: 180 &amp;mgr; m (~ 150um bei älteren Tieren) </li></ul> Scheiben bei 37 º C für 1 Stunde (einschließlich Schneiden Zeit), dh, lassen in Bad 15-20 Minuten nach dem Schneiden. Pipetten: Presyn: 3,5-4,5 MOhm auf den ersten, 3,0 bis 3,5 MOhm, wenn sich sicherer fühlen Verwenden Sie puller Handbuch Einstellungen vornehmen. Verwenden Sie Delay Feature, denn Glas ist dick (Verzögerungen von 200 oder 1 Arbeitsplatz) Glass Art: Borosilikatglas, Standard-Wand, kein Filament Außendurchmesser: 2,0 mm Innendurchmesser 1,16 mm Länge: 7,5 cm

methods for patch clamp capacitance recordings from the calyx

Wir zeigen die grundlegenden Techniken für die präsynaptische Patch-Clamp-Aufzeichnung am Kelch der Held, ein zentralen Nervensystem von Säugetieren Nervenendigung. Elektrische Aufnahmen aus den präsynaptischen Terminal ermöglichen die Messung der Aktionspotentiale, Calcium-Kanal-Ströme, Vesikelfusion (Exozytose) und anschließende Membran-Aufnahme (Endozytose). Die Fusion von Vesikeln mit Neurotransmitter bewirkt, dass die Vesikelmembran an die Zellmembran der Kelch hinzugefügt werden. Dieser Anstieg in Höhe von Zellmembran ist eine Erhöhung der Kapazität gemessen. Die anschließende Reduktion der Kapazität zeigt Endozytose, den Prozess der Membran die Aufnahme oder Streichung aus dem Kelch Membran. Endozytose, ist notwendig, um die Struktur der Kelch zu halten und es ist auch notwendig, um Vesikel, die mit Neurotransmitter für zukünftige Exozytose Ereignisse gefüllt werden bilden. Kapazität Aufnahmen am Kelch von Held haben es möglich gemacht, um direkt und schnell messen vesikuläre Freisetzung und anschließende Endozytose in einem Säuger-ZNS Nervenendigung. Darüber hinaus können die entsprechenden postsynaptischen Aktivität gleichzeitig mit gepaart Aufnahmen gemessen werden. So ein vollständiges Bild von der präsynaptischen und postsynaptischen elektrische Aktivität an einem zentralen Nervensystems Synapse ist erreichbar mit diesem Präparat. Hier werden die Methoden zur Scheibe Vorbereitung, morphologische Merkmale zur Identifizierung von Kelchen der Held, grundlegende Patch-Clamp-Techniken und Beispiele der Kapazität Aufnahmen Exozytose und Endozytose Maßnahme.

Watch this Scientific Journal Video about Methods for Patch Clamp Capacitance Recordings from the Calyx at JoVE.com

Methods for Patch Clamp Capacitance Recordings from the Calyx

Methoden für die Patch-Clamp Kapazität Recordings aus dem Kelch

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

methods-for-patch-clamp-capacitance-recordings-from-the-calyx

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K Aufrufe.  National Institute of Health. 

Serie: Methods for Patch Clamp Capacitance Recordings from the Calyx

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 ...

Forschung

Biologie

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pressure-Polieren Pipetten für verbesserte Patch-Clamp Recording

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

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Elektrophysiologischen Methoden für die Aufzeichnung Synaptic Potentials aus dem NMJ von Drosophila-Larven

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Methoden zur Untersuchung des Zebrafisch Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

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 ...

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.

Exploring arteriellen glatten Muskelzellen KV7 Kalium Kanal Funktion mit Patch-Clamp-Elektrophysiologie und Druck Myographie

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.

Riesen-Liposomen-Herstellung für Imaging und Patch-Clamp-Elektrophysiologie

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.

Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme

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.

Rekonstitution der ein Transmembranprotein, das spannungsgesteuerten Ionenkanal, KvAP, in Riesen unilamellare Vesikel für Mikroskopie und Patch-Clamp-Studien

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.

Implementieren der Patch-Clamp und Live-Fluoreszenzmikroskopie, um funktionelle Eigenschaften von frisch isolierten PKD Epithel-Monitor

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