패치 클램프 설정 : 광학 : 좋은 세포를 찾으려면, 그리고 그것의 지위를 확립하기 위해 광학은 매우 중요합니다. 날카로운 모서리, 소프트 보이는 세포 : 세포의 명확한 이미지를보십시오. 첨부 꽃받침 잘 MNTB 교장 세포 찾기 위해 전체 필드를 검사합니다. 다른 각도에서 꽃받침의 복수를 찾는 요리를 회전합니다. 처음에는 그것은 풍선 모양의 터미널에서 연습을 확인합니다. 슬라이스의 표면에 세포를 선택하거나, 2 차 전지 레이어 인치 피펫과 세포의 첫번째 3분의 1 차를 대상으로 Calyxes을 파악하면 : 이중 막 (그림 1 참조)을 찾아, 그것이 꽃받침 있는지 확인 수 있도록 여러 각도에서 볼 수있는 슬라이스 접시를 돌린다. <img src="/files/ftp_upload/old/244_2008_8_22_2/244_Figure-01.png" alt="그림 1" width="298" height="159" /> 그림 1 칼리의 깊이 : 칼리는 표면에이거나 가능한 표면에 가까이해야합니다. 패치를 사용할 수 표면 : 확인하기 위해 현미경에 사용 미세 초점 노프 방법 &quot;깊이 / 폭&quot;꽃받침입니다. 예를 들어, 잘 집중할 전체 차례 ~ 10 %는 당신에게에 패치 충분한 면적 이상을 제공할 것입니다. presynaptic 세포에 실링 : 연결된 세포에 도착하려면, 피펫의 POS 압력 (~ 0.15psi)에 의한 세포의 표면에 &quot;작은&quot;변형을보고, 아래와 전역, 피펫을 실행하는 조작하는 사람을 사용하여, 심지어로 필요한 경우와 같은 좋은 변형을 얻기 위해 presyn 단말기의 표면은, 다음, 압력을 릴리스하고 흡입을 적용합니다. PULSE, 데이터 수집 프로그램 : 처음 사용하기 전에 버퍼 크기를 증가하고, 언제든지 샘플링 용량을 필요로합니다. 수정된 매크로 매번로드해야합니다. 잠재 액체 융티온 : Presyn 녹화 솔루션 (부정적 11mV)입니다 - 11mV. Presyn : Cslow = ~ 15 PF presyn을위한 RS 광고 : 10 μsec, 65 % (더 많은 보상 수도 있습니다 가능한 W / 아웃 oscillations) 레코딩 용량하기 전에 테스트 현재 휴식 (10 뮤직 비디오, 10 밀리초 점프)이 휴식이 monoexponential되고 있는지 여부, 즉 presyn 용어가 하나의 구획으로 모델 수 있는지 여부. 필터 : 빠른 구성 요소를 놓쳤다하지 않도록 휴식을 측정하는 동안 30 kHz에서 10 kHz에서 (각각 베셀 필터 1, 2)에서 설정합니다. 미니 녹음하는 동안, Xinsheng 10 kHz에서와 5kHz를 사용합니다. LGW 우리가 미니의 상승 시간을 측정할 수 있도록 30 kHz에서에서만 필터 1 세트를 사용하는 나에게 물었다. 파일 이름 : 7 자리 mm 한달입니다 000mmxx (또는 달과 연도)과 XX는 핸드폰 번호입니다. 솔루션 라인 청소 : 0.1 M HCL 20 ML와 실험 끝에 청소 린스 200 ML ddH2O 다음. 외부 솔루션을 재활용, 펌프 속도는 20-30 RPM입니다. (목욕 챔버에서 돌아오는 솔루션 눈금 실린더에 다시 흐르고). 충분한 흡입을 보장하기 위해 3 나가는 라인을 설정합니다. 피펫 Presynaptic 녹화 솔루션 : ~ 310 밀리 Osmolar Presyn 솔루션 실험 기간 동안 R - 액세스 증가를 방지하기 위해 ~ 310 mOsm해야합니다. Presyn 피펫 솔루션은 postsyn 피펫 용액 (Presyn은 은퇴한에 대한 ATP 및 GTP 수 있습니다.) 다릅니다. TTX - 티 솔루션 (CA 전류를 분리) : <ul><li> 솔루션 블록 K - 채널에서 차 같이, 세포에, 실링, 또는 패치 이후에 적용하려고하고 세포를 depolarizes. 이것은 세포에 좋은하지 않습니다. 같은 이유로, 그것은 TTX가 적용된 후 슬라이스를 다시 사용할 수 없을 수도 있습니다. </li><li> 세포 또는 장기 탈분극의 효과를 확인하기 위해 슬라이스의 첫 번째와 마지막 응답을 비교합니다. </li><li> 효과가 빨리되도록 TTX는 1 μm의 대신에 0.5 μm의에 사용됩니다. 솔루션을 변경한 후에는 더 적은 시간을 기다릴 필요. </li></ul> 절단 조각 Vibratome 설정 : <ul><li> 70Hz 수평 진동 </li><li> 진폭 : 1.0 mm </li><li> 꽃받침 조각과를 깎다가 전달 0.1 달리 두께가 0.01-0.02 mm / 초 속도 : 180 μm의를 (~ 150이전 동물 μm의) </li></ul> 조각은 1 시간 (절삭 시간을 포함), 즉 15~20분 절단 후 욕조에서 떠날에 대한 37 º C에서 알을 품다. Pipettes : Presyn : 처음 MΩ 3.5-4.5, 3.0-3.5 MΩ 더 자신감 설정을 설정하는 풀러 설명서를 사용합니다. 유리 두께 때문에 지연 기능을 사용하여 (200 또는 1 일의 지연) 유리 유형 : Borosilicate, 표준 벽, 아니 필라멘트 외경 : 2.0 mm 내경 1.16 mm 길이 : 7.5 cm

methods for patch clamp capacitance recordings from the calyx

우리는 헬드, 포유류의 중추 신경의 신경 단말기의 꽃받침에 presynaptic 패치 클램프 녹음을위한 기본 기술을 보여줍니다. presynaptic 터미널에서 전기 녹음 작업 잠재력, 칼슘 채널 전류, 소포의 융합 (exocytosis)와 후속 막 이해 (endocytosis)의 측정을 수 있습니다. 신경 전달 물질을 포함한 vesicles의 융합이 꽃받침의 세포막에 추가하는 소포 막 발생합니다. 세포막의 금액이 증가 커패시턴스의 증가로 측정됩니다. 커패시턴스의 후속 감소 endocytosis, 멤브레인 이해 또는 꽃받침 막에서 제거하는 과정을 나타냅니다. Endocytosis은 꽃받침의 구조를 유지하기 위해 필요하고 또한 앞으로 exocytosis 이벤트에 대한 신경 전달 물질로 가득합니다 vesicles를 형성하는 것이 필요합니다. 개최의 꽃받침에 커패시턴스 레코딩은 가능한 직접 빠르게 소낭성의 릴리스와 포유 동물 CNS 신경 터미널에서 후속 endocytosis을 측정하기 위하여 만들었습니다. 또한, 해당 postsynaptic 활동이 동시에 결합하여 레코딩을 사용하여 측정할 수 있습니다. 따라서 중추 신경계의 버렸네에서 presynaptic 및 postsynaptic 전기 활동의 완전한 그림이 준비를 사용하여 달성됩니다. 여기, 슬라이스 준비, 개최, 기본 패치 클램핑 기술 꽃받침의 복수의 식별 형태학의 기능 및 exocytosis와 endocytosis를 측정 커패시턴스 레코딩의 예제에 대한 방법은 제공됩니다.

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

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

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

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Biology

12.6K 조회수.  National Institute of Health. 

비디오: 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.

Xenopus Oocytes의 표현 이온 채널 패치 클램프 녹음

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

연구

생물학

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.

향상된 패치 - 클램프 녹음 압력 연마 Pipettes

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.

Drosophila의 애벌레의 NMJ에서 시냅틱 후보를 녹음 Electrophysiological 방법

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.

Zebrafish 상악 바벌의 연구 방법

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.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 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 ...

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.

패치 클램프 전기 생리학 및 압력 Myography를 사용하여 간선 평활근 Kv7 칼륨 채널 기능을 탐색

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.

이미징 및 패치 클램프 전기 생리학에 대한 거대한 리포좀 준비

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.

1 채널의 세포 부착 된 패치 클램프 녹음

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.

횡단 단백질의 재구성, 현미경 및 패치 클램프 연구 거대한 단일 층 소포에 전압 게이트 이온 채널, KvAP,

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.

패치 클램프 및 라이브 형광 현미경을 구현 갓 고립 PKD 상피 세포의 기능적 특성을 모니터링하려면

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