Culture de cellules Les cellules peuvent être cultivées en utilisant des techniques établies, mais doit être plaqué sur des lamelles en verre # 1 enduit d&#39;un adhésif cellulaires (comme la polylysine, polyornithine ou de la laminine) pour empêcher les cellules de se détacher ni bouger pendant des expériences d&#39;imagerie. Solutions Des expériences d&#39;imagerie calcique peut être effectuée en utilisant une variété de solutions physiologiques, y compris les milieux de culture cellulaire. Il est important, cependant, de s&#39;assurer que les solutions sont libres de rouge de phénol, ce qui augmente considérablement le fond fluorescent. Nous utilisons la solution Tyrodes, qui se fait facilement et imite le liquide céphalorachidien, et nous le compléter avec du sérum bovin 0,1% d&#39;albumine. Nous utilisons une dépolarisation avec mM de chlorure de potassium 60-90 pour activer les canaux calciques voltage dépendants et thapsigargine 1 uM (1 mM dans le DMSO stocks) ou 2 pm ionomycine (1 mM dans le DMSO stocks) pour activer magasin exploité canaux CRAC. Il est souvent pratique pour enlever le calcium de la solution extracellulaire de montrer que les élévations de calcium sont dus à l&#39;afflux de calcium. Lors de la suppression de calcium, il est nécessaire de maintenir la concentration totale des cations bivalents (Mg 2 + et Ca 2 +) constant. En cas de remplacement du potassium pour le sodium, il est nécessaire de maintenir l&#39;équilibre osmotique. Solutions Tyrodes: <table border="1"><tbody><tr><td></td><td> Faible taux de potassium 2 mM de Ca 2 + Tyrodes (mM) </td><td> Faible taux de potassium 0 Ca 2 + Tyrodes (mM) </td><td> Haute de potassium 2 mM de Ca 2 + Tyrodes (mM) </td><td> Haute de potassium 0 Ca 2 + Tyrodes (mM) </td></tr><tr><td> NaCl </td><td> 129 </td><td> 129 </td><td> 5 </td><td> 5 </td></tr><tr><td> KCl </td><td> 5 </td><td> 5 </td><td> 129 </td><td> 129 </td></tr><tr><td> CaCl2 </td><td> 2 </td><td> 0 </td><td> 2 </td><td> 0 </td></tr><tr><td> MgCl2 </td><td> 1 </td><td> 3 </td><td> 1 </td><td> 3 </td></tr><tr><td> Le glucose </td><td> 30 </td><td> 30 </td><td> 30 </td><td> 30 </td></tr><tr><td> Hepes </td><td> 25 </td><td> 25 </td><td> 25 </td><td> 25 </td></tr></tbody></table> Ajuster le pH à 7,4 avec NaOH Chargement de la teinture de calcium Fura-2 Nous chargeons les cellules avec acétoxy-méthyl-ester Fura-2 (Fura-2 AM), qui diffuse à travers la membrane cellulaire et est de-estérifiés par des estérases cellulaires pour produire Fura-2 sans acide. Les paramètres exacts pour Fura-2 de chargement varient énormément selon les types de cellules. Nous recommandons de tester diverses conditions en préparant des solutions de chargement de plusieurs contenant une concentration multiple de Fura-2 fait rage 1-4 uM, l&#39;incubation des cellules dans la solution de chargement pour une variété de fois à partir de 15 minutes à 2 heures et le test du chargement à température ambiante et à 37 deg. Un protocole simplifié pour les neurones corticaux est donnée ci-dessous: <ol><li> Tout d&#39;abord, préparer le 1 mM Fura-2 heures en ajoutant 50 pl stocks de DMSO dans un flacon 50μg disponibles chez Invitrogen. Il est important d&#39;utiliser le DMSO sec emballé sous azote et il est nécessaire d&#39;éliminer le DMSO avec une aiguille en perforant le septum pour empêcher l&#39;hydratation du DMSO. Après la préparation de la solution Fura-deux heures le conserver dans un endroit sombre et sec. Fura-deux heures dans le DMSO est stable à température ambiante pendant 24 heures et est stable à - 20 degrés dans un récipient sec pendant plusieurs mois. </li><li> Aliquote de 2 ml de milieux de culture dans un tube de 15 ml conique, chaude à 37 degrés. et ajouter des 2μl Fura-2 heures pour produire un stock de 1 uM Fura-2 heures solution. Vortex la solution vigoureusement pendant 1 minute. </li><li> Transférer la solution de chargement pour une boîte de culture tissulaire 35 mm et le transfert de la lamelle avec les cellules dans le plat. </li><li> Incuber les neurones à 37 degrés pendant 30 minutes dans un endroit sombre incubateur. Temps d&#39;incubation de la précision. </li><li> Préparer un plat de 35 mm contenant 2 ml de milieux de culture tissulaire, sans Fura-2 heures. Enlever les lamelles de la solution de chargement et de place dans le nouveau plat. </li><li> Montez la lamelle sur la chambre d&#39;imagerie. Enlever les lamelles de l&#39;antenne de 35 mm et rapidement monter sur la chambre en prenant soin de prévenir l&#39;assèchement des cellules. Nous utilisons une chambre d&#39;imagerie fabriqué par Warner Instruments qui permet une lamelle de 10mm contenant les cellules à être monté sur le fond et une lamelle deuxième à être monté sur le haut formant un sandwich. Les deux lamelles sont sécurisés avec de la graisse à vide à la chambre et deux tubes à chaque extrémité de la chambre de permettre la perfusion de solutions à travers la chambre. La ligne d&#39;entrée est reliée à une seringue et la ligne de sortie est reliée à un puits qui est vidé par une ligne d&#39;aspiration relié à un piège à vide. </li></ol> Le microscope Nous utilisons une inversé Nikon Eclipse TE2000-U microscope équipé d&#39;une lampe au xénon à arc (Sutter Instruments), une étape automatisée (Ludl), une roue de filtre d&#39;excitation (Ludl), et une charge de refroidissement couple appareil (CCD) (Hamamatsu Orka II). Le microscope est contrôlé par un com Macintoshordinateur exécutant le logiciel Lab ouvert (Improvisation). Plusieurs autres logiciels sont disponibles pour l&#39;imagerie ratiométrique. Pour l&#39;imagerie que nous utilisons 40, 60 ou 100x Nikon objectifs Fluor immersion dans l&#39;huile avec un NA dépassant pas 1,2. Imagerie Protocole <ol><li> Calibrer la platine du microscope. </li><li> Charge Tyrodes solution dans la ligne d&#39;entrée en prenant soin d&#39;éviter la formation de bulles d&#39;air. </li><li> Connectez la chambre pour les lignes de perfusion et perfuser Tyrodes solution à travers la chambre une fois de plus, en prenant soin d&#39;éviter la formation de bulles dans la chambre </li><li> Déposer une goutte d&#39;huile sur l&#39;objectif, placez la chambre sur la platine du microscope et de se concentrer sur les cellules en utilisant la lumière transmise. </li><li> Examinez la fluorescence des cellules en utilisant un éclairage à 340 et à 380 nm en utilisant les oculaires. Les cellules au repos doivent être sombres et lumineux à 340 à 380. En général, les cellules ne doivent pas être éclairée par une lumière UV pendant plus de 10 ou 15 secondes et l&#39;intensité de la lumière d&#39;excitation doit être réduite avec une densité neutre filtre pour empêcher la phototoxicité. </li><li> Examiner les cellules en utilisant l&#39;appareil et réglez le gain et l&#39;exposition de la caméra pour générer une image qui est proche de la saturation (mais pas saturé) lorsqu&#39;il est éclairé à 380 nm et bien en dessous de la saturation lorsqu&#39;il est éclairé à 340 nm. Gardez l&#39;exposition en dessous de 200ms si possible. Une fois fixé, ne modifiez pas le gain de la caméra ou l&#39;exposition à moins que vous prévoyez aussi modifier l&#39;arrière-plan, Rmin et Rmax (voir ci-dessous). </li><li> Recueillir une image à chaque longueur d&#39;onde et de l&#39;utilisation de la région d&#39;intérêt (ROI) outil pour mesurer l&#39;intensité de l&#39;expérience dans une variété d&#39;endroits dans les images pour chaque longueur d&#39;onde. </li><li> Moyenne des valeurs de fond et entrez les valeurs de fond dans les endroits appropriés dans le programme d&#39;imagerie. La valeur de fond sera soustrait de chaque pixel dans le domaine. </li><li> Recueillir une image ratio de pratiques et d&#39;adapter les valeurs seuils pour chaque longueur d&#39;onde pour générer un rapport d&#39;image qui inclut uniquement les cellules et non le fond et qui n&#39;est pas bruyant dans la région près des bords des cellules. </li><li> Réglez la valeur de ratio minimum (Rmin) à environ 10% en dessous du ratio le plus bas de n&#39;importe quelle cellule dans le domaine. </li><li> Réglez le RMax à environ 12 fois le Rmin. Ne changez pas le rmin ou RMax entre les expériences que vous envisagez de comparer car il fera la comparaison difficile. Nous avons rarement changer le Rmin et les valeurs Rmax sur notre banc d&#39;imagerie. </li><li> Utilisez la scène automatisé de trouver les champs que vous prévoyez d&#39;image et enregistrer l&#39;emplacement de chaque champ en utilisant le logiciel. En général, nous recueillons cinq champs de vue lors d&#39;une expérience. Le stade de mouvements automatiques à un champ, recueille un rapport d&#39;image et se déplace sur le champ suivant. </li><li> Régler l&#39;intervalle de temps-faute pour recueillir des images entre 0,1 et 10 secondes d&#39;intervalle, selon le type de signaux que vous vous attendez à voir. </li><li> Démarrer l&#39;expérience. </li><li> Lors du test du système d&#39;imagerie du calcium, il est souvent utile d&#39;utiliser des stimuli tels que Tyrodes élevé en potassium (65 mM de KCl) ou ionomycine (2 pm) qui va provoquer une élévation du calcium intracellulaire et extracellulaire de calcium solution gratuite qui va (contenant les tampons de calcium, comme l&#39;EGTA et BAPTA ) qui permettra de réduire la concentration de calcium. </li></ol> Analyse Une fois l&#39;expérience terminée, vous voulez convertir l&#39;ensemble des images dans le rapport de mesures du calcium time-lapse pour des cellules individuelles ou des régions d&#39;intérêt dans les cellules. Pour ce faire: <ol><li> Utilisez la région de l&#39;outil d&#39;intérêt (ROI) pour définir les zones de l&#39;image dans laquelle vous voulez mesurer le calcium. Il est généralement utile d&#39;avoir au moins un retour sur investissement qui couvre le corps cellulaire de la cellule. Lors de la définition du ROI, il est utile de lire le film sur les images pour vérifier que les cellules ne se déplacent pas pendant le cours de l&#39;expérience. </li><li> Utilisez le logiciel pour collecter des mesures de rapport time-lapse pour chaque ROI de chaque image. </li><li> Importer les mesures du rapport dans un programme d&#39;analyse. Vous aurez besoin de voir les traces de calcium pour des cellules spécifiques ou ROIs, moyenne ou plusieurs cellules ROIs et convertir les mesures du rapport à la valeur intracellulaire de calcium. Nous utilisons un ensemble de macros écrites en Pro Igor qui nous permettent de faire toutes ces tâches d&#39;analyse commune, mais d&#39;autres programmes comme Excel, bien que moins pratique peut également être utilisé. </li><li> L&#39;équation [Ca] = (RR min) / (R max-R) Sf * Kd peut être utilisé pour convertir les valeurs du rapport Fura-2 pour les concentrations intracellulaires de calcium. [Ca] est la concentration de calcium, R est le Fura-2 340/380 ratio, Rmin et Rmax sont les ratios 340/380 en l&#39;absence de calcium ou en présence d&#39;une concentration saturante de calcium, respectivement, et Sf * Kd est le produit de la Kd de Fura-2 (environ 120 nm à température ambiante) et une valeur d&#39;échelle. Pour mesurer R Min, Max et R * Sf Kd, il est nécessaire d&#39;effectuer soit un in vivo ou in vitro une dans l&#39;étalonnage. Lors de la calibration in vivo requiert unecombinaison de patch clamp et d&#39;imagerie calcique, qui peut être compliqué, mais est également très précis. Lors de la calibration in vitro peut être fait en utilisant un home-made chambre de calibration qui se compose de deux lamelles séparées par une entretoise fine lamelle de générer une fine couche de solution dans laquelle effectuer l&#39;étalonnage. Le moyen le plus commode de mesurer des valeurs Fura-2 rapport en fonction des concentrations de calcium est d&#39;utiliser un kit de calibration qui contiennent plusieurs solutions de calcium tamponné et Fura-2 sans acide. Ceux-ci peuvent être obtenus auprès de Invitrogen (Molecular Probes) et contiennent des instructions précises pour l&#39;utilisation. </li></ol>

<ol>
	<li>Grynkiewicz, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J. Biol. Chem. 260, 3440-3450 (1985).</li><li>Lewis, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> Cell Regul. 1, 99-112 (1989).</li><li>Dolmetsch, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+between+intracellular+Ca2++stores+and+depletion-activated+Ca2++channels+generates+[Ca2+]i+oscillations+in+T+lymphocytes.">Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes.</a> J Gen Physiol. 103, 365-388 (1994).</li></ol>

Dans cette présentation, nous avons franchi toutes les étapes pour effectuer l&#39;imagerie calcique utilisant Fura-2 heures. Nous avons utilisé les neurones corticaux comme un modèle, mais l&#39;imagerie calcique peuvent être utilisés pour mesurer le calcium intracellulaire en temps réel dans une variété de cellules. Lorsque vous effectuez cette procédure, il est important d&#39;utiliser des lamelles en verre au lieu du plastique, le plastique est souvent fluorescentes à des longueurs d&#39;onde ultraviolettes, ce qui rend l&#39...

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calcium imaging of cortical neurons using fura-2 am

Imagerie calcique est une technique courante qui est utile pour mesurer les signaux de calcium dans les cellules cultivées. Les techniques d&#39;imagerie calcique profiter de colorants indicateurs de calcium, qui sont BAPTA basé molécules organiques qui changent leurs propriétés spectrales en réponse à la fixation d&#39;ions Ca2 +. Colorants indicateurs de calcium entrent dans deux catégories, le ratio-métrique colorants comme la Fura-2 et indo-1 et une seule longueur d&#39;onde, comme les teintures fluo-4. Ratio-métriques colorants changer soit leur excitation ou de leurs spectres d&#39;émission en réponse au calcium, ce qui permet la concentration de calcium intracellulaire d&#39;être déterminé à partir du ratio d&#39;émission de fluorescence ou d&#39;excitation à des longueurs d&#39;onde distinctes. Le principal avantage de l&#39;utilisation de ratio-métrique par rapport aux sondes colorants seule longueur d&#39;onde, c&#39;est que le rapport signal est indépendante de la concentration de colorant, de l&#39;intensité d&#39;éclairage, et la longueur du chemin optique permettant la concentration de calcium intracellulaire à être déterminée indépendamment de ces artefacts. Un des indicateurs de calcium la plus commune est Fura-2, qui a un pic d&#39;émission à 505 nm et change son pic d&#39;excitation de 340 nm à 380 nm en réponse à la fixation du calcium. Nous décrivons ici l&#39;utilisation de la Fura-2 pour mesurer les élévations de calcium intracellulaire dans les neurones et autres cellules excitables.

Watch this Scientific Journal Video about Calcium Imaging of Cortical Neurons using Fura-2 AM at JoVE.com

Calcium Imaging of Cortical Neurons using Fura-2 AM

Signaux calciques jouent un rôle clé dans de nombreux processus cellulaires, y compris l&#39;expression des gènes, la survie et la différenciation. Ici, nous montrent comment effectuer l&#39;imagerie calcique utilisant Fura-2 heures. Imagerie calcique est un outil précieux pour étudier la régulation du calcium intracellulaire en temps réel et de sa réglementation des cascades de signalisation.

Imagerie calcique des neurones corticaux utilisant Fura-2 heures

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

calcium-imaging-of-cortical-neurons-using-fura-2-am

Research

JoVE Journal

Biology

60.6K vues.  Stanford University. Signaux calciques jouent un rôle clé dans de nombreux processus cellulaires, y compris l'expression des gènes, la survie et la différenciation. Ici, nous montrent comment effectuer l'imagerie calcique utilisant Fura-2 heures. Imagerie calcique est un outil précieux pour étudier la régulation du calcium intracellulaire en temps réel et de sa réglementation des cascades de signalisation.

Vidéo: Calcium Imaging of Cortical Neurons using Fura-2 AM

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Préparation E18 neurones de rat corticale compartimentation dans un dispositif microfluidique

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Recherche

Biologie

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Imaging calcium dans la couche 2 / 3 de souris

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagerie en temps réel du noyau dense de vésicules cultures primaires neurones de l&#39;hippocampe

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

L&#39;imagerie in vivo de Deep couches corticales utilisant un microprisme

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

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.

Basées sur la fluorescence de mesure de l&#39;entrée du calcium magasin fonctionnant dans des cellules vivantes: à partir de cellules cancéreuses en culture de fibre musculaire squelettique

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

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Imagerie directe du calcium ER avec Targeted-estérase colorant induit Chargement (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Imagerie confocale à base de FRET mesure des fluctuations de calcium à l&#39;intérieur du réticulum sarcoplasmique de Cultured cellules musculaires lisses Utilisation

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Concurrent EEG and Functional MRI Recording and Integration Analysis for Dynamic Cortical Activity Imaging

Electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are two of the fundamental noninvasive methods for identifying brain activity. Multimodal methods have sought to combine the high temporal resolution of EEG with the spatial precision of fMRI, but the complexity of this approach is currently in need of improvement. The protocol presented here describes the recently developed spatiotemporal fMRI-constrained EEG source imaging method, which seeks to rectify source biases and improve EEG-fMRI source localization through the dynamic recruitment of fMRI sub-regions. The process begins with the collection of multimodal data from concurrent EEG and fMRI scans, the generation of 3D cortical models, and independent EEG and fMRI processing. The processed fMRI activation maps are then split into multiple priors, according to their location and surrounding area. These are taken as priors in a two-level hierarchical Bayesian algorithm for EEG source localization. For each window of interest (defined by the operator), specific segments of the fMRI activation map will be identified as active to optimize a parameter known as model evidence. These will be used as soft constraints on the identified cortical activity, increasing the specificity of the multimodal imaging method by reducing cross-talk and avoiding erroneous activity in other conditionally active fMRI regions. The method generates cortical maps of activity and time-courses, which may be taken as final results, or used as a basis for further analyses (analyses of correlation, causation, etc.) While the method is somewhat limited by its modalities (it will not find EEG-invisible sources), it is broadly compatible with most major processing software, and is suitable for most neuroimaging studies.

An EEG-fMRI multimodal imaging method, known as the spatiotemporal fMRI-constrained EEG source imaging method, is described here. The presented method employs conditionally-active fMRI sub-maps, or priors, to guide EEG source localization in a manner that improves spatial specificity and limits erroneous results.

Simultanées EEG et IRM fonctionnelle enregistrement et analyse d’intégration pour l’imagerie dynamique activité corticale

Electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) are two of the fundamental noninvasive methods for identifying brain ...

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 ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagerie de la dynamique du calcium dans les sous-populations de cellules d'islet pancréatique de souris

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...