L&#39;induction de SRD adoptifs S&#39;il vous plaît voir l&#39;article de jove: <a href="/index/Details.stp?ID=325">induction et la surveillance des parents adoptifs hypersensibilité de type retardé chez les rats</a> Christine Beeton, George K. Chandy Département de physiologie et de biophysique, Université de Californie, Irvine Expériences J. visualisés, Numéro 8 Ici, nous avons modifié ce protocole en utilisant un antigène (ovalbumine), conjugué au Texas-Red dans un rapport 1:1 avec l&#39;antigène non marqué pour permettre la visualisation des phagocytes cellules présentatrices d&#39;antigène. Récolte tissu de l&#39;oreille <ol><li> Euthanasier les rats à la fois du point souhaité par les procédures décrites dans le protocole d&#39;animaux approuvés. Ici, nous utilisons une surdose de l&#39;anesthésique, l&#39;isoflurane. Ceci est fait dans un récipient fermé situé à l&#39;intérieur d&#39;une hotte bien ventilée. Assurez-vous que l&#39;animal est mort soit par décapitation de l&#39;ouverture de la cavité thoracique et la coupe du coeur. </li><li> Avec des ciseaux grande retirer l&#39;oreille avec une seule coupe, au plus près du corps de l&#39;animal que possible (figure 1). <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> Figure 1. Suppression de l&#39;oreille chez le rat </li><li> Placez l&#39;oreille dans un tube de 15 ml conique contenant 10 ml de ~ glacée 1x PBS ou RPMI-1640. Conserver les échantillons de tissu sur la glace jusqu&#39;à ce que vous êtes prêt à préparer les tissus pour l&#39;imagerie. </li><li> Image le tissu le plus tôt possible; nous trouvons, cependant, que 1 à 2 heures sur la glace n&#39;a pas d&#39;effet discernable sur la motilité cellulaire dans les tissus par rapport aux tissus récoltés et imagé immédiatement. </li></ol> Préparation des tissus pour l&#39;imagerie Retrait I. couche épidermique et dermique * Note: La couche de derme profond doit être gardée intacte. Si fait correctement les gros vaisseaux sanguins restera intact et le cartilage de l&#39;oreille ne sera pas exposée. C&#39;est une technique difficile, surtout quand il n&#39;ya pas d&#39;inflammation (les conditions de contrôle) et il est utile d&#39;utiliser un microscope à dissection. <ol><li> Placez les deux le tissu et les médias du tube conique dans un plat de 10 cm de culture de tissu. </li><li> Avec les mains gantées maintenir l&#39;oreille du rat doucement à la pointe de l&#39;oreille (distale) avec la face dorsale de l&#39;oreille vers le haut (figure 2). <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> Figure 2. La fourrure Coupez l&#39;oreille </li><li> Avec votre pouce sur le dessus de l&#39;oreille, soutenue le tissu tout en utilisant la pointe d&#39;fermés Dumont # 5 ciseaux pour séparer le derme du derme profond. Alternativement, si le bord de l&#39;oreille a un surplomb de la peau / derme profond, ce qui peut être saisie avec une pince et tire doucement pour séparer ce à partir des tissus en dessous (figure 3). <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> Figure 3. Suppression de la partie supérieure du derme </li><li> Une fois une petite zone de peau a été séparée du tissu sous-jacent, répétez les étapes de séparation utilisant alternativement des ciseaux pour couper entre les couches dermiques et une pince pour enlever délicatement et retirez la peau délogé. </li><li> Exposer un 5 x 5 mm ou plus grande surface de tissu de l&#39;oreille qui est au moins 3 mm sur le site initial de l&#39;injection d&#39;antigène (figure 4). <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> Figure 4. Tissu exposé pour l&#39;imagerie </li></ol> II. Sécurisation du tissu à l&#39;étape d&#39;imagerie <ol><li> Couper une lamelle en plastique d&#39;une taille légèrement plus grande que les tissus préparés. </li><li> Étendre une mince couche de 3M ™ Vetbond tissus sûre de la colle sur la diapositive. </li><li> Saisir le coin du tissu préparé (où vous ne sont pas susceptibles d&#39;image), retirer du média, et tamponnez la partie inférieure du tissu sur du papier lentille ou Kym essuyer pour enlever l&#39;excès des médias. </li><li> Toucher le bord du tissu à la diapositive de la colle couverts (les guérisons Vetbond immédiatement sur tissu humide). Étirez le tissu à plat, avec l&#39;extrémité opposée étant le dernier à toucher la lame (figure 5) <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> Figure 5. Mont mouchoir pour vous couvrir de glissement </li><li> Cure ™, Vetbond en inversant le tissu échantillon / glisser et en le plongeant dans un réservoir de médias tels que les tissus et les diapositives sont face vers le bas et à peu près à un angle de 45 degrés. Tenir le tissu à l&#39;envers à un angle permet d&#39;éviter l&#39;excès de colle de durcir à la surface des tissus exposés. Colle sur la surface du tissu va bloquer le laser (figure 6). <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> Figure 6. Cure tissus Vetbond glue par trempage dans les médias Remarque: étapes 2-5 peuvent prendre un peu de pratique pour maîtriser. Essayez de pratiquer avec des morceaux de tissus jetés ou extra avant de tenter pour la première fois. </li><li> Tamponnez une petite quantité de graisse silicone sur le bas de la diapositive. </li><li> Placer la lame dans la chambre de perfusion. Assurez-vous que les médias perfusion coule, 37 ° C et oxygéné avant d&#39;ajouter des tissus. Appuyez doucement sur les bords de la lame faisant un joint avec la graisse silicone entre le fond de la chambre de perfusion et de la lamelle en plastique. </li></ol> III. Deux-Photon Imaging <ol><li> Utiliser champ lumineux lumière transmise, se concentrer sur le haut du tissu. Eteignez toutes les lumières. </li><li> Allumez le laser, PMT tel que requis par votre système de microscope multi-photons. </li><li> Deuxième génération d&#39;harmoniques (SHG) dans les protéines fibreuses hautement ordonné (collagène et myosine) produit un signal à la moitié de la longueur d&#39;onde du laser d&#39;excitation. Nous utilisons une excitation à 900 nm, conduisant à SHG à 450 nm, qui peuvent être visualisées en utilisant les &quot;bleus&quot; canal du microscope. SHG génération est un non-linéaires (à deux photons) processus, et fournit ainsi une optique &quot;sectionner&quot; effet similaire à celui de l&#39;excitation à deux photons de fluorescence. Le rendement de pointe de SHG se produit lorsque les fibrilles sont perpendiculaires au laser, tandis que les fibrilles qui s&#39;exécutent en parallèle ne sont pas visualisées. 1 </li><li> Repérez une zone d&#39;imagerie bonne avec beaucoup de cellules, définissez votre zone d&#39;acquisition de telle sorte que la majorité des cellules sont au centre d&#39;imagerie et de commencer. Remarque: Dans les réactions DTH, les cellules T et des infiltrats de cellules d&#39;autres se trouvent souvent à se concentrer ensemble tandis que d&#39;autres zones sont dépourvues d&#39;infiltrer les cellules 2. Par conséquent, il peut prendre un certain temps à chercher le tissu pour trouver la meilleure zone de l&#39;image. </li><li> Vérifier la viabilité des cellules en évaluant la vitesse et de la polarité cellulaire lors de l&#39;imagerie. Utilisez la puissance plus bas au laser qui fournit une qualité d&#39;image acceptable pour assurer un minimum de photo-dommages. </li><li> Swap dans une préparation de nouveaux tissus, au besoin. </li></ol>

<ol>
	<li>Koo, G. C., 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=Blockade+of+the+voltage-gated+potassium+channel+Kv1.3+inhibits+immune+responses+in+vivo.">Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.</a> J Immunol. 158, 5120-5128 (1997).</li><li>Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+activation+and+T+lymphocyte+infiltration+in+allergen-induced+late+phase+skin+reactions+and+classical+delayed-type+hypersensitivity.">Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity.</a> J Immunol. 147, 816-822 (1991).</li><li>Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoaggressive+effector+T+cells+in+the+course+of+experimental+autoimmune+encephalomyelitis+visualized+in+the+light+of+two-photon+microscopy.">Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.</a> J Neuroimmunol. 191, 86-97 (2007).</li><li>Kawakami, N., 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=Live+imaging+of+effector+cell+trafficking+and+autoantigen+recognition+within+the+unfolding+autoimmune+encephalomyelitis+lesion.">Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.</a> J Exp Med. 201, 1805-1814 (2005).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Matheu, M. P. B., A, C. G. a. r. c. i. a., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Imaging+of+Effector/Memory+T+Cells+During+DTH+and+Suppression+by+Kv1.3+Channel+Block.">In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block.</a> Immunity. , (2008).</li></ol>

imaging effector memory t cells in the ear after induction of adoptive dth

Type de l&#39;hypersensibilité retardée (DTH) est une réaction immunitaire dans laquelle les principaux acteurs sont CCR7 - effecteur / lymphocytes T mémoire. Ici, nous démontrons une méthode pour induire et d&#39;enregistrer les progrès d&#39;une réaction d&#39;hypersensibilité retardée dans l&#39;oreille de rat. Elle est suivie par une démonstration de la préparation du tissu de l&#39;oreille chez le rat pour l&#39;imagerie à deux photons de l&#39;CCR7 - la réponse des cellules effectrices / T mémoire. Un SRD adoptifs est induite par l&#39;injection intrapéritonéale de GFP-étiquetée spécifiques de l&#39;OVA CCR7 - ligne de cellules effectrices / T mémoire (Beeton, C J. expériences visualisées, numéro 8). Les cellules sont ensuite autorisés à s&#39;équilibrer dans le rat pendant 48 heures avant l&#39;épreuve par l&#39;injection d&#39;une oreille avec une solution saline (contrôle oreille) et l&#39;autre avec un mélange 1:1 de conjugués d&#39;ovules et d&#39;ovules au Texas-Red (Ova-TR) pour permettre la visualisation des résidents cellules présentatrices d&#39;antigène. Nous décrivons une méthode de préparation des tissus utiles pour l&#39;imagerie de la motilité des cellules dans la couche profonde cutanée durant une réponse immunitaire, en conjonction avec la visualisation des fibres de collagène par la génération de second harmonique. Tissu de l&#39;oreille est coupé en carrés de 5 mm x 5 (légèrement plus grand, c&#39;est mieux) et monté sur des lamelles en plastique à l&#39;aide Vetbond ™, qui sont ensuite fixés avec de la graisse de silicone dans une chambre d&#39;imagerie et perfusées par l&#39;oxygène-bulles moyennes de culture de tissus à 37 ° C .

Watch this Scientific Journal Video about Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH at JoVE.com

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Ici nous démontrons une méthode pour induire et d&#39;enregistrer les progrès d&#39;un retard de type hypersensibilité (SRD) réaction de l&#39;oreille chez le rat. Elle est suivie par une démonstration de la préparation du tissu de l&#39;oreille chez le rat pour l&#39;imagerie à deux photons de la réponse cellulaire effecteur / T mémoire.

Imagerie Les cellules T effectrices de mémoire dans l&#39;oreille après induction de SRD adoptifs

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

imaging-effector-memory-t-cells-ear-after-induction-adoptive

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Biology

10.2K vues.  University of California, Irvine (UCI). Ici nous démontrons une méthode pour induire et d'enregistrer les progrès d'un retard de type hypersensibilité (SRD) réaction de l'oreille chez le rat. Elle est suivie par une démonstration de la préparation du tissu de l'oreille chez le rat pour l'imagerie à deux photons de la réponse cellulaire effecteur / T mémoire.

Vidéo: Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

L&#39;induction et le suivi des actifs d&#39;hypersensibilité de type retardé (DTH) chez les rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

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Biologie

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

L&#39;induction et la surveillance des parents adoptifs hypersensibilité de type retardé chez les rats

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Test clonogénique: cellules adhérentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imagerie des récepteurs d&#39;estrogènes α chez le rat artérioles Pial aide d&#39;un microscope numérique immunofluorescence

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagerie Ca locale<sup&gt; 2+</sup&gt; Signaux de cellules de mammifères cultivées

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Récupération de fluorescence après Photoblanchiment du jaune Fluorescent Protein tag p62 en Structures induites ressemblant à Aggresome

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Dissection efficace et culture des cellules épithéliales rétiniennes primaires de pigment de souris

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...