Les auteurs tiennent à remercier Glen Slocum (Medical College of Wisconsin) et Colleen A. Lavin (Nikon Instruments, Inc.) pour une excellente assistance technique avec des expériences de microscopie. Gregory Blass est reconnu pour la relecture critique du manuscrit. Cette recherche a été financée par les Instituts nationaux de la santé HL108880 de subvention et American Diabetes Association accorder 1-15-BS-172 (AS), et Ben J. Lipps bourse de recherche de l&#39;American Society of Nephrology (DVI).

L&#39;utilisation et le bien-être des animaux devraient adhérer à la NIH Guide pour le soin et l&#39;utilisation des animaux de laboratoire suivant les protocoles examinés et approuvés par le Comité de protection des animaux et l&#39;utilisation institutionnelle (IACUC). 1. Kidney Flush <ol><li> Utiliser 8 à 12 semaines vieux rat mâle (suggéré est une souche Sprague Dawley, mais d&#39;autres souches d&#39;âge et sexe différent peuvent être utilisés avec les modifications appropriées). </li><li> Anesthésier les animaux selon le mode opératoire autorisé par le protocole IACUC; surveiller la profondeur de l&#39;anesthésie et d&#39;inspecter l&#39;animal. La description détaillée de l&#39;opération à effectuer dans 1.3 à 1.8 peuvent être trouvés dans Ilatovskaya 7 et al. </li><li> Après une anesthésie adéquate, placer l&#39;animal sur une table chirurgicale à température contrôlée, faire une incision médiane de l&#39;abdomen (jusqu&#39;à 3 pouces de longueur), et de découvrir la veine cave et l&#39;aorte. </li><li> Insérez ligature autour les cœliaques et mésentériques supérieure artères et les abdominauxceux au-dessus de l&#39;aorte; ne pas ligaturer. </li><li> Blunt disséquer l&#39;aorte abdominale dessous des artères rénales, et placez deux ligatures autour de lui, mais ne ligaturer, puis serrer l&#39;aorte au-dessus des ligatures et attacher le fil inférieur. </li><li> Cathétériser l&#39;aorte avec une tubulure PE50 polyéthylène (attaché à une pompe à seringue remplie de PBS) au-dessous de la pince et fixer le cathéter à la seconde ligature; retirer la pince, tourner sur la pompe, et ligaturer l&#39;aorte et des artères mésentériques avec coeliaques. Vite incision dans la veine rénale pour soulager la pression. </li><li> Infuser l&#39;aorte avec le PBS pré-refroidi pendant 2 ou 3 minutes à une vitesse de 6 ml / min. </li><li> Arrêtez la perfusion, accises et décapsuler 7 les reins, et les mettre sur la glace dans la solution PBS. Euthanasier l&#39;animal selon un protocole approuvé par le IACUC. </li></ol> 2. Isolement des glomérules Rat <ol><li> Préparer 30 ml de solution fraîche de 5% de BSA dans du milieu RPMI 1640. </li><li> En utilisant une lame de rasoir et des ciseaux,isoler le cortex des deux reins, puis émincer jusqu&#39;à homogénéité. Cette procédure a été décrite précédemment 7. </li><li> Appuyez sur le tissu haché lors de l&#39;étape précédente à travers un tamis en acier inoxydable de 100 mesh (pré-trempés dans 5% de BSA en solution / RPMI) à l&#39;aide d&#39;une spatule. Recueillir l&#39;écoulement et par la force de gravité permettent l&#39;écoulement de passer à travers un tamis 140 mesh. </li><li> Filtrer la accréditives recueillies auprès des 140 tamis en utilisant un tamis à mailles de 200 pré-imprégné, jeter le filtrat, et laver le haut du tamis avec 10 - 15 ml de la solution préparée BSA / RPMI pour recueillir les glomérules que les sédiments sur le tamis. </li><li> Mettre la solution de BSA / RPMI contenant glomérules sur de la glace dans un tube de 15 ml et laisser le sédiment de glomérules au fond du tube pour un maximum de 20 min. Le concentré de glomérules sur le fond du tube sera clairement visible. Retirer la solution en excès, en laissant environ 2 ml dans le tube. </li></ol> 3. Single-channel patch-clamp Electrophysiology <ol><li> Préparer 5 x 5 mm éclats de verre de couverture en les enduisant d&#39;MW 70.000 - 150.000 poly-ʟ-lysine, et laisser sécher. Utilisez environ 30 pi de 0,01% solution filtrée stérile dans l&#39;eau par verre de couverture. </li><li> Réchauffez les solutions expérimentales à RT et remplir la chambre de patch-clamp et la pipette. Pour les canaux de TRPC surveillance, utiliser une solution de bain, en mm: 126 NaCl, CaCl2 1, 10 HEPES, 2 MgCl 2, 10 glucose, pH 7,4; pipette: 126 NaCl, CaCl2 1,5, HEPES 10, 10 glucose; pH 7,4. <ol><li> Ajouter inhibiteurs à la solution de la pipette pour bloquer l&#39;activité de canaux endogènes, qui ne sont pas pertinentes pour les études (recommandées sont les suivantes: 100 uM acide niflumique ou DIDS (pour bloquer Ca 2+ activés par canaux Cl -), 10 mM TEA (pour inhiber la grande conductance Ca 2 + - K + dépendante canal), 10 nM ibériotoxine (pour bloquer les Ca2 + canaux K + activés par), 10 uM nicardipine (pour bloquer Ca de type N2+ canaux)) directement avant l&#39;expérience de patch-clamp. </li></ol></li><li> Mélanger doucement la solution contenant glomérules, et ensuite appliquer environ 50 pi de celui-ci à la couverture enduite éclats de verre poly-ʟ-lysine. Laissez les glomérules attachent pendant environ 5 min. </li><li> Déplacer les éclats de verre avec glomérules à la chambre de patch-clamp pré-remplies avec la solution de bain; perfuser la chambre à un débit de 3 ml / min pendant 1 min à assurer l&#39;élimination des glomérules seules. </li><li> Mener une expérience de patch-clamp conventionnel dans un mode cellule attachée 7. Avec une pipette en verre (7 à 10 MQ de résistance à la pipette) forment un joint d&#39;étanchéité à haute résistance entre une pipette et une membrane de podocytes par application d&#39;une aspiration douce (une pipette fixée à un podocytes sur la surface du glomérule isolé est représenté sur la figure 2, sur la gauche). </li><li> Pour les mesures ci-joint cellules passe-bas, les courants à 300 Hz par un filtre à huit pôles Bessel. </li><li> Usoi isolé glomérules dans les expériences de patch-clamp pour un maximum de 4 - 6 h. Gardez la fraction stock glomérules sur la glace. </li></ol> 4. Ratiométrique confocale de fluorescence mesures de la concentration intracellulaire de calcium dans les podocytes <ol><li> Placez 500 pi de la fraction de glomérules (décrit à 2,5) dans 0,5 ml tube conique et ajouter des colorants de calcium Fura rouges, AM et Fluo-4, AM. Utilisez 2 mm et 1 mm actions concentrations de Fura-Rouge, AM et Fluo-4, AM, respectivement (conserver à -20 ° C, dissous dans du DMSO) et utiliser 2,5 pi de chaque colorant pendant 500 pi de fraction de glomérules. Immédiatement après l&#39;ajout des colorants couvrir le tube de papier d&#39;aluminium. </li><li> Placer les tubes sur un agitateur rotatif pendant au moins 20 minutes jusqu&#39;à une heure à température ambiante. Remarque: Les agents pharmacologiques peuvent être ajoutés au cours de cette étape. </li><li> Préparer des lamelles de verre, les couvrir avec de la poly-ʟ-lysine et de permettre un séchage en utilisant ensemble de plaque chauffée à 70 ° C. </li><li> Une fois le chargement des colorants de calcium est complete, applique 100 pl de solution contenant les glomérules des lamelles couvre-objet enduites de poly-lysine-ʟ et qu&#39;ils adhèrent à la surface pendant environ 5 min. Monter les lamelles de glomérules inscrits dans une chambre de formation d&#39;image, et perfuser avec la solution de bain (contenant (en mM): 145 NaCl, 4,5 KCl, 2 MgCl2, 10 HEPES, pH 7,35) à un débit de 3 ml / min pour supprimer les glomérules seules et les colorants restants. </li><li> Régler la confocal à balayage laser à une longueur d&#39;onde d&#39;excitation de 488 nm et de filtres d&#39;émission (525/25 et 650/25 nm pour Fluo-4 et Fura-Rouge, respectivement). Réglez le logiciel d&#39;imagerie à une fréquence et la résolution souhaitée. </li><li> Trouver les glomérules en clair et puis tourner sur la détection du signal de fluorescence. Ajuster l&#39;intensité du laser pour chaque colorant pour éviter la saturation du signal. Choisir le plan focal avec les podocytes qui sont directement attachés à la vitre. Ceci minimise l&#39;effet provoqué par la contraction d&#39;un glomérule en réponse à médicamentapplication. Double-vérifier que le glomérule de choix est bien fixé à la vitre; </li><li> Lancer l&#39;imagerie du plan focal de choix (prendre 512 x 512 images avec la fréquence fixée à 4 secondes de visualiser Ca 2+ rapide des changements transitoires. Utilisez un / NA 1.4 ou objectif 60X similaire image haute résolution pour), appliquer médicaments d&#39;intérêt, et enregistrer la réponse. <ol><li> Sélectionner un plan focal souhaité (le plus près de la surface du verre, donc, pour assurer l&#39;imagerie de podocytes sur la surface du glomérule). Vérifiez l&#39;intensité de fluorescence sur le Fluo4 et canaux FuraRed, et assurez-vous que le glomérule est clairement visible dans fond clair. </li><li> Lancer imagerie. Avant l&#39;application de toutes les drogues, fiche d&#39;au moins 1 min de fluorescence de base pour vous assurer que le signal est stable (il n&#39;y a pas de pointes soudaines ou évanouissement du signal). </li><li> Appliquer médicaments souhaités à l&#39;aide d&#39;une micro-pipette; soyez prudent et veiller à la drogue était capable de bien diffuser et atteindre le glomerUlus. Mélanger la solution de bain en douceur si nécessaire, suivre le plan focal choisi et vérifier qu&#39;il ne bougeait pas de mise au point en raison de la demande de drogue. </li><li> Enregistrer les variations de l&#39;intensité de fluorescence pour les signaux Fluo4 et FuraRed. Assurez-vous que l&#39;enregistrement est assez long, en attendant que le signal atteint le niveau du plateau ou atteint un pic, puis retourne à la ligne de base. Effectuer le changement de la solution ou l&#39;addition de tous les autres médicaments si nécessaire. </li><li> Arrêtez l&#39;enregistrement, et enregistrer le fichier dans le format natif du logiciel. </li></ol></li></ol> 5. Analyse d&#39;image pour les mesures de calcium <ol><li> Effectuer l&#39;analyse d&#39;image avec le logiciel ImageJ ouverte équipée avec le plugin utilitaire ND qui permet d&#39;importer des images dans le format natif ND2. </li><li> Importez la séquence d&#39;images; assurez-vous de séparer les canaux et utiliser un mode HyperStack en niveaux de gris. </li><li> Suivez l&#39;→ Outils → ROI chemin Analyser de gestionnaire dans le logiciel ImageJ à oStylo une fenêtre du gestionnaire de ROI. Sélectionnez plusieurs régions d&#39;intérêt (podocytes) en utilisant un outil de sélection ovale et le &quot;(t) Ajouter&quot; fonction dans le Gestionnaire de ROI. Comme la dernière ROI, sélectionnez la zone à l&#39;arrière-plan; enregistrer les ROI sélectionnés (Plus → Enregistrer). </li><li> Mettez en surbrillance la fenêtre contenant le canal sélectionné pour analyses. Utilisez la fonction multi More → Mesurer dans le Gestionnaire de ROI, cochez la case &quot;une ligne par tranche» dans le dialogue qui sort, puis cliquez sur OK. Les résultats seront affichés dans le format qui peut être mis en place dans la fenêtre des résultats: entrer dans les options de menu Résultats → Set Mesures, sélectionnez &quot;Valeur moyenne grise» et de calculer les valeurs d&#39;intensité de pixel pour chaque ROI, qui sera affiché dans la fenêtre Résultats dans des colonnes distinctes pour chaque ROI. </li><li> Copiez les valeurs d&#39;intensité de ROI mesurées pour chaque canal (Fluo-4 et Fura-Rouge) dans le logiciel d&#39;analyse de données pratique; valeurs d&#39;intensité de fond Soustraire de chaque datun point. </li><li> Pour chaque point de temps de calculer le rapport des intensités du Fluo-4 à canaux Fura rouges. Terrain scatter / ligne change de point de temps de la Ca transitoire pour chaque ROI. Calculer les valeurs moyennes / SE pour glomérules sélectionnés. Remarque: la colonne de temps devrait être fixé en fonction de la fréquence d&#39;imagerie sélectionné à 4,5. </li></ol> 6. Calculs intracellulaires de calcium de concentration en utilisant Fluo-4 Fluorescence Signal <ol><li> Prélever un échantillon des glomérules et exécuter le protocole expérimental jusqu&#39;à l&#39;étape 4.7. Après l&#39;enregistrement de la fluorescence de fond ajouter ionomycine (concentration finale dans la chambre de bain doit être de 10 uM) à la solution de bain, et de l&#39;augmentation de l&#39;intensité de fluorescence fiche. Une fois l&#39;intensité atteint son maximum et la décroissance commence, ajouter MnCl2 (concentration finale doit être de 5 mm) pour étancher la fluorescence 8. </li><li> Analyser les données obtenues en 6.1. Copier les valeurs d&#39;intensité de ROI Fluo-4 signal obtenues selonle protocole décrit à 5/1 à 5/5 par un logiciel d&#39;analyse préféré. </li><li> Calculer la concentration en calcium intracellulaire (en nM) dans le podocyte correspondant à la ROI en utilisant une formule: <img alt="Equation 1" src="/files/ftp_upload/52850/52850equation1.jpg" /> où K d est une dissociation prédéterminée constante de Fluo-4 (345 nM), F est l&#39;intensité au point de temps que vous êtes le calcul de la concentration en calcium de (niveau de référence), et F min et F max sont les valeurs d&#39;intensité au point de charge maximale de calcium (après application ionomycine) et après extinction de la fluorescence (avec MnCl 2), respectivement (voir Figure 3). </li></ol>

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Les auteurs ont rien à révéler.

L&#39;approche décrite ici permet l&#39;analyse de manipulation de calcium par les podocytes des glomérules chez les rongeurs. Cette technique permet l&#39;application de patch-clamp électrophysiologie à canal unique et imagerie confocale à fluorescence ratiométrique. Cependant, les deux approches peuvent être utilisés séparément, sur leur propre. Le protocole proposé comporte plusieurs étapes relativement simples, y compris 1) Kidney Flush; 2) l&#39;isolement des glomérules par tamisage différentiel; 3) e...

Les reins maintiennent l&#39;équilibre homéostatique pour diverses substances et de réguler le volume sanguin d&#39;une manière qui détermine la pression sanguine totale. Des perturbations dans la filtration rénale, la réabsorption ou la sécrétion d&#39;entraîner ou d&#39;accompagner des états pathologiques, allant de l&#39;hyper ou hypotension pour mettre fin à l&#39;insuffisance rénale qui nécessite éventuellement une transplantation rénale. L&#39;unité de filtration rénale (glomérule) se compose de trois couches - l&#39;endothélium capillaire, la membrane basale et une couche mono-cellulaire des cellules épithéliales - podocytes, qui jouent un rôle majeur dans le maintien de l&#39;intégrité et de la fonction 1 fente diaphragme. Dysfonction dans le filtre glomérulaire provoque une perte urinaire à perméabilité sélective de macromolécules, telles que la protéinurie. Différents agents peuvent affecter la structure des podocytes et leurs procédés de pied, qui déterminent l&#39;intégrité de la barrière de filtration de glomérules. Les podocytes sont impliqués dans le maintien de la glomfonction de filtration Eruli. Il a été établi que la manipulation de calcium impropre à l&#39;podocytes conduit à une lésion de la cellule et joue un rôle important dans la progression de diverses formes de néphropathies 2,3. Par conséquent, le développement d&#39;un modèle qui permet de mesure directe des changements de concentration de calcium intracellulaire sera déterminante pour les études de la fonction des podocytes. Glomérules isolés ont été précédemment utilisés dans de nombreuses études, y compris une mesure du coefficient de réflexion albumine change 4 et l&#39;évaluation des courants cellulaires intégraux dans les cellules entières électrophysiologiques mesures de patch-clamp 5,6. Dans le présent article, nous décrivons le protocole qui permet au chercheur de mesurer intracellulaires changements de concentration de calcium en réponse aux demandes d&#39;agents pharmacologiques, estimer les niveaux de calcium dans les cellules basales, et d&#39;évaluer l&#39;activité des particuliers canaux calciques. Mesures de la concentration de calcium Ratometric et patch-clamp electrophysiology ont été utilisés pour déterminer respectivement des changements dans la concentration de calcium intracellulaire au sein de l&#39;activité de canal et podocytes.

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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.

Ici, nous avons abordé le problème de la mesure des changements de courte durée dans les niveaux de calcium dans les podocytes. La figure 1 montre une représentation schématique du protocole expérimental conçu pour effectuer l&#39;imagerie haute résolution fluorescence confocale en temps réel et des enregistrements individuels de l&#39;activité des canaux ioniques dans les podocytes de la fraîchement glomérules isolés de rongeurs. Brièvement, après le rat est anesthésié, les reins doive...

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

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

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.

Single-channel Analyse et Imagerie de calcium dans les podocytes des glomérules fraîchement isolés

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

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

Research

JoVE Journal

Biology

10.9K vues.  Medical College of Wisconsin. 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. 

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

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagerie fonctionnelle de la graisse brune chez les souris avec FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Recherche

Biologie

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Évaluation de calcium Sparks dans intactes les fibres musculaires squelettiques

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

Quantifier simple microvaisseaux perméabilité d&#39;isolement de poumon de rat Préparation du sang perfusé

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

La mesure de la perméabilité à l&#39;eau Coefficient osmotique (P<sub&gt; F</sub&gt;) Des cellules sphériques: protoplastes de plantes isolées comme un exemple

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.

Mise en œuvre de patch clamp en direct et microscopie de fluorescence pour surveiller propriétés fonctionnelles des fraîchement isolés PKD épithélium

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La perfusé Technique du rein de souris isolé

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Analyse de la fonction des cellules bêta à l’aide d’imagerie calcique résolution unicellulaire dans les îlots de poisson-zèbre

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Mesure simultanée de Calcium intracellulaire et le potentiel de Membrane dans l’endothélium cérébral de souris fraîchement isolées et intacte

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...