ونود أن نشكر الدكتور Claycomb لتوفير خلايا HL-1 و بروتوكول مفصل صيانة. كما نود أن نشكر السيدة ايلينا كاريو والدكتور سيث Robia على المساعدة التي قدموها مع توليد الفيلم الخلية والسيد بيت كارون لمساعدته مع جعل بعض الملحقات لدينا مزدوج نظام رسم الخرائط البصرية القناة. وأيد هذا العمل من قبل جمعية القلب الأمريكية (10GRNT3770030 و12GRNT12050478 إلى XA)، المعاهد الوطنية للصحة (HL113640 إلى XA)، وصندوق لويولا جامعة بحوث التنمية (لXA).

<ol>
	<li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li><li>Salama, G., Hwang, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.]. 12 (Unit 12 17), (2009).</li><li>Laurita, K. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain+inhibition+prevents+pacing-induced+cellular+remodeling+in+a+HL-1+myocyte+model+for+atrial+fibrillation.">Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation.</a> Cardiovasc Res. 62 (3), 521-528 (2004).</li><li>Umapathy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogram+fractionation+in+murine+HL-1+atrial+monolayer+model.">Electrogram fractionation in murine HL-1 atrial monolayer model.</a> Heart Rhythm. 5 (7), 1029-1035 (2008).</li><li>Fast, V. G., Cheek, E. 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No conflicts of interest declared.

توضح هذه المقالة الجوانب الرئيسية لرسم الخرائط البصرية في تربيتها HL-1 الأذيني العضلية أحادي الطبقة ملطخة الكالسيوم والجهد الأصباغ الفلورية الحساسة. يشمل ذلك زراعة والأمثل HL-1 أحادي الطبقة الخلية، والإعداد للمعدات رسم الخرائط، ورسم خرائط أحادي الطبقة المثقفة، وتحلي...

والكالسيوم فريد (كاليفورنيا) والجهد (V م) المزدوج رسم الخرائط الضوئية قنوات تقنية 1-5 يبرز بوصفه أداة فعالة لتسجيل وقت واحد V م والكالسيوم إشارات في كل من قلوب سليمة والطبقات الوحيدة الخلية مثقف. هذا الأسلوب يجعل من الممكن الحصول على معلومات قوية بشأن العلاقة بين العابرين الكالسيوم وإمكانات العمل لفهم أفضل للآليات الكهربية الكامنة وراء عدم انتظام ضربات القلب. وقد أثبتت الطبقات الوحيدة خلايا مستنبتة لتكون نموذجا الخلوي المفيد دراسة الكهربية القلب وآلية الكامنة وراء عدم انتظام ضربات القلب. 4،6-8 HL-1 الخلايا العضلية هي الأذيني خط ثقافة تتميز بشكل جيد. خلايا HL-1 أيضا الحفاظ على النمط الجيني متباينة بشكل فريد والنمط الظاهري الذي يتضمن المورفولوجي، الكهربية، والخصائص الدوائية من myocytes الأذيني الكبار. هذه الخلايا تعبر عن الجينات والبروتينات القلب، بما في ذلك importaالإقليم الشمالي القنوات الأيونية القلب (أي L- وقنوات الكالسيوم T-نوع) 9 و الإسوية ناضجة من البروتينات مقلص الساركومير توجد عادة في myocytes الأذيني الكبار مثل الآخرين ولقد ذكرت سابقا. 10-13 وبالإضافة إلى ذلك، يمكن HL-1 خلايا يكون مثقف لتشكيل 2-الأبعاد (2-D) العضلية أحادي الطبقة. وهكذا، فإن مزايا استخدام مثقف الطبقات الوحيدة HL-1 ما يلي: 1) أقل نسبيا من حيث التكلفة وأسهل للحفاظ على خط الثقافة العضلية من عزل وزراعة myocytes حديثي الولادة الأولية؛ 2) أحادي الطبقة متموجة من الخلايا يقلل من التعقيدات الهيكلية التي تنجم عن هيكل 3-D من القلب؛ 3) يمكن أحادي الطبقة خلية القضاء على التدخل من التليف الخلالي الذي يحدث في قلب سليمة. وهذا يمكن أن تستخدم لتشريح وظائف الكهربية محددة من مجموعة من myocytes دون تدخل من الخلايا الليفية ومصفوفة الخلالي. 4) تقييم عواقب وظيفية من التلاعب الدوائي أو الوراثي في ​​طريق مسدودالطبقات الوحيدة الخلية بناءه من جديد يمكن أن يتحقق على نحو فعال. لذلك، أصبحت HL-1 خلايا نموذجا الخلوية المستخدمة على نطاق واسع لدراسة الجوانب المختلفة لعلم وظائف الأعضاء العضلية وكذلك الأنشطة الكهربائية غير الطبيعية الناجمة عن سرعة. 13-16 ومع ذلك، لا بد من معالجة خاصة ورعاية للثقافة الطبقات الوحيدة الخلية الصحيحة التي تستجيب للخارجية سرعة الكهربائية للدراسات رسم الخرائط البصرية. وبالإضافة إلى ذلك، قد المزدوج الفلورسنت الإجراء صبغ تلطيخ تلف بسهولة على سلامة مثقف الطبقات الوحيدة الخلية متموجة. وهكذا، فقد كان أداء V م / CA قناة مزدوجة رسم الخرائط البصرية في تربيتها HL-1 الطبقات الوحيدة تحديا كبيرا. والهدف من هذه الطريقة هو توفير الخطوات الرئيسية لأداء بنجاح المزدوج رسم الخرائط الضوئية القنوات في تربيتها HL-1 الطبقات الوحيدة. نحن هنا قدمت تفاصيل واسعة على بروتوكول الأمثل لإعداد أحادي الطبقة HL-1 خلية، المزدوج رسم الخرائط البصرية قناة أحادي الطبقة خلايا مستنبتة، ومعالجة البيانات ورسم الخرائط.

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voltage and calcium dual channel optical mapping of cultured hl-1 atrial myocyte monolayer

وقد ثبت رسم الخرائط الضوئية لتكون تقنية قيمة للكشف عن النشاط الكهربائي في القلب على حد سواء سليمة خارج الحي قلوب والطبقات الوحيدة العضلية مثقف. وقد استخدمت على نطاق واسع خلايا HL-1 كنموذج الخلوي 2-الأبعاد لدراسة الجوانب المختلفة لعلم وظائف الأعضاء القلب. ومع ذلك، فقد كان تحديا كبيرا لخريطة بصريا الكالسيوم (الكالسيوم) العابرين وإمكانات العمل في وقت واحد من نفس مجال الرؤية في تربيتها HL-1 خلية الأذيني أحادي الطبقة. هذا هو لأنه مطلوب معالجة خاصة والرعاية لإعداد الخلايا السليمة التي يمكن أن يتم القبض كهربائيا وتعيين بصريا. لذلك، قمنا بتطوير بروتوكول العمل الأمثل لالمزدوج رسم الخرائط البصرية القناة. في هذه المخطوطة، التي وصفناها بالتفصيل كيفية تنفيذ القناة التجربة رسم الخرائط الضوئية المزدوجة. هذا البروتوكول هو أداة مفيدة لتعزيز مفهوم العمل نشر المحتملين وحركية الكالسيوم في التنمية عدم انتظام ضربات القلب.

<html> ومتموجة أحادي الطبقة المثقفة المعارض إيقاع جوهري منتظم كما هو موضح في الفيلم 1. نحن ثم يقوم V م / كا المزدوج رسم الخرائط البصرية القناة في متموجة بشكل كامل HL-1 أحادي الطبقة. الشكل 1A معارض سبيل المثال آثار V م وإشارات الكالسيوم من تسجيل وا...

Watch this Scientific Journal Video about Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer at JoVE.com

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

الجهد والكالسيوم مزدوجة القناة البصرية رسم خرائط مثقف HL-1 الرجفان العضلية واحد رقائق

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

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10.1K Views.  Loyola University Chicago. This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Video: Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

العناية والتنظيف السليم للمجهر

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

وعلى النقيض من مرحلة التدخل التفاضلية المجهري التباين (DIC)

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

رسم الخرائط البصرية القلوب الجرذ Langendorff - perfused

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

البصرية تسجيل النشاط الكهربائي في غينيا الخنازير المعوية باستخدام شبكات الجهد حساسة ملونات

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Photoconversion منقى من البروتينات ونيون المزدوجة التحقيق تسليط الضوء البصري في الخلايا الحية

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

مضان القائم على القياس من دخول الكالسيوم المتجر الذي يعمل في الخلايا الحية: من خلية السرطان مثقف على عضلة

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

العزلة وتسجيلات قناة كيلو فولت في الفئران Cardiomyocytes الأذيني البطيني و

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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.

إعادة تشكيل البروتين عبر الغشاء، وايون القناة بوابات الجهد، KvAP، إلى العملاق Unilamellar الحويصلات للفحص المجهري وتصحيح الدراسات المشبك

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

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

على قناة واحدة وتحليل الكالسيوم التصوير في Podocytes من الكبيبات المعزولة حديثا

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

رسم الخرائط البصرية البينية العضلية شبكية كاليفورنيا<sup&gt; 2+</sup&gt; وإمكانية الغشاء في قلب الأرنب Langendorff perfused-

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...