Induzione di DTH adottivi Si prega di consultare l&#39;articolo jove: <a href="/index/Details.stp?ID=325">induzione e monitoraggio del adottivi ipersensibilità ritardata nei ratti</a> Christine Beeton, George K. Chandy Dipartimento di Fisiologia e Biofisica, University of California, Irvine Esperimenti J. visualizzati, Numero 8 Qui, abbiamo modificato questo protocollo usando antigene (ovoalbumina), coniugata con Texas-Red in un rapporto 1:1 con l&#39;antigene senza etichetta per consentire la visualizzazione di fagocitaria cellule presentanti l&#39;antigene. Orecchio tessuto Harvest <ol><li> Euthanize il ratto al momento desiderato-punto per le procedure descritte nel protocollo animali approvato. Qui, usiamo una dose eccessiva di anestetico, isoflurano. Questo viene fatto in un contenitore chiuso all&#39;interno di una cappa ben ventilata. Assicurarsi che l&#39;animale è morto da una decapitazione del aprendo la cavità toracica e tagliando il cuore. </li><li> Utilizzando le forbici grandi rimuovere l&#39;orecchio con un unico taglio, più vicino al corpo dell&#39;animale possibile (Figura 1). <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> Figura 1. La rimozione dell&#39;orecchio ratto </li><li> Posizionare l&#39;orecchio in un tubo da 15 ml contenente circa 10 ml di ghiacciata 1x PBS o RPMI-1640. Mantenere i campioni di tessuto sul ghiaccio fino al momento di preparare il tessuto per l&#39;imaging. </li><li> L&#39;immagine del tessuto il più presto possibile, troviamo, però, che 1 o 2 ore sul ghiaccio non ha alcun effetto distinguibile sulla motilità delle cellule nei tessuti rispetto al tessuto raccolto e ripreso immediatamente. </li></ol> Preparazione dei tessuti per l&#39;imaging Rimozione I. strato epidermico e dermico * Nota: Lo strato dermico profondo deve essere mantenuta intatta. Se fatto correttamente grandi vasi sanguigni rimarrà intatto e la cartilagine dell&#39;orecchio non saranno esposti. Questa è una tecnica difficile, soprattutto quando non c&#39;è l&#39;infiammazione (condizioni di controllo) ed è utile usare un microscopio da dissezione. <ol><li> Luogo sia il tessuto e dei media dal tubo conico in un piatto di coltura di tessuti 10 cm. </li><li> Con le mani guantate tenere l&#39;orecchio ratto delicatamente sulla punta dell&#39;orecchio (distale) con la parte dorsale del l&#39;orecchio rivolto verso l&#39;alto (Figura 2). <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> Figura 2. Pelliccia tagliare l&#39;orecchio </li><li> Con il pollice sulla parte superiore dell&#39;orecchio, stabile il tessuto mentre si usa la punta di chiusura Dumont # 5 forbici per separare il derma dal derma profondo. In alternativa, se il bordo del padiglione auricolare ha una sporgenza della pelle / derma profondo, questo può essere afferrato con una pinza e delicatamente tirato a separare questo dal tessuto sottostante (Figura 3). <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> Figura 3. La rimozione del derma superiore </li><li> Una volta che una piccola zona di pelle è stata separata dal tessuto sottostante, ripetere la procedura di separazione in alternativa usare le forbici per tagliare tra gli strati del derma e pinze per rimuovere delicatamente e rimuovere la pelle sloggiato. </li><li> Esporre un 5 x 5 mm o maggiore superficie di tessuto orecchio che è di almeno 3 mm dal sito iniziale di iniezione antigene (Figura 4). <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> Figura 4. Tessuti esposti per l&#39;imaging </li></ol> II. Protezione del tessuto allo stage Imaging <ol><li> Tagliare un pezzo di plastica di copertura a una dimensione leggermente più grande rispetto al tessuto preparato. </li><li> Stendere uno strato sottile di 3M ™ Vetbond tessuto-safe colla tutta la diapositiva. </li><li> Afferrare l&#39;angolo del tessuto preparato (dove non sono suscettibili di immagine), rimuovere dai media, e tamponare la parte inferiore del tessuto su carta lenti o Kym pulire per rimuovere il supporto in eccesso. </li><li> Toccare il bordo del tessuto alla diapositiva colla coperto (le cure Vetbond immediatamente sul tessuto bagnato). Tratto delicatamente il tessuto piatto, con l&#39;estremità opposta è l&#39;ultimo a toccare la diapositiva (Figura 5) <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> Figura 5. Monte dei tessuti per fodera </li><li> Curare l&#39;™ Vetbond invertendo il campione di tessuto / slide e immersione in un serbatoio di media in modo tale che il tessuto e far scorrere sono a faccia in giù e più o meno ad un angolo di 45 gradi. Tenendo premuto il tessuto a testa in giù in un angolo aiuta a prevenire la colla in eccesso da curare sulla superficie del tessuto esposto. Colla sulla superficie del tessuto blocca il laser (Figura 6). <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> Figura 6. Cure vetbond tessuto glue per immersione nei media Nota: 2-5 passaggi possono prendere un po &#39;di pratica per padroneggiare. Prova pratica con pezzi di tessuto scartati o più prima di provare per la prima volta. </li><li> Tamponare una piccola quantità di grasso al silicone sul fondo della diapositiva. </li><li> Mettere il vetrino nella camera di perfusione. Assicurarsi che il supporto perfusione è scorrevole, 37 ° C e ossigenata prima di aggiungere dei tessuti. Premere delicatamente sul bordo del vetrino fare un sigillo con il grasso al silicone tra il fondo della camera di perfusione e la polizza di copertura in plastica. </li></ol> III. Due-Photon Imaging <ol><li> Utilizzando campo chiaro luce trasmessa, messa a fuoco sulla parte superiore del tessuto. Spegnere tutte le luci. </li><li> Accendere il laser, PMT, come richiesto dal sistema multi-fotone microscopio. </li><li> Generazione di seconda armonica (SHG) in altamente ordinato proteine ​​fibrose (collagene e miosina) produce un segnale a metà della lunghezza d&#39;onda del laser di eccitazione. Noi usiamo eccitazione a 900 nm, portando a SHG a 450 nm che può essere visualizzato utilizzando il canale &quot;blu&quot; del microscopio. SHG generazione è un non-lineare (a due fotoni) di processo, e fornisce quindi un effetto ottico &quot;sezionare&quot; simile a quella di eccitazione a due fotoni di fluorescenza. L&#39;efficienza di picco di SHG si verifica quando le fibrille sono perpendicolari al laser, mentre le fibrille che corrono in parallelo non verrà visualizzato 1. </li><li> Individuare una buona zona di imaging con abbondanza di celle, impostare l&#39;area di tale acquisizione che la maggior parte delle cellule si trovano nel centro e iniziare imaging. Nota: Nelle reazioni DTH, le cellule T e cellule infiltrati altri si trovano spesso a concentrarsi insieme mentre altre zone sono prive di infiltrare cellule 2. Pertanto, si può richiedere un certo tempo a cercare i tessuti per trovare la zona migliore per immagine. </li><li> Verificare la presenza di vitalità cellulare, valutando la velocità e la polarità delle cellule durante l&#39;imaging. Utilizzare il minor potenza laser che offre una qualità dell&#39;immagine accettabile per garantire la minima foto-danni. </li><li> Swap in un preparato nuovo tessuto, se necessario. </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

Ipersensibilità di tipo ritardato (DTH) è una reazione immunitaria in cui i protagonisti sono CCR7 - effettrici / linfociti T memoria. Qui, dimostriamo un metodo per indurre il progresso e la registrazione di una reazione DTH nell&#39;orecchio ratto. Questa è seguita da una dimostrazione della preparazione di tessuto di ratto orecchio per l&#39;imaging a due fotoni del CCR7 - effettrici / risposta delle cellule T memoria. Un DTH adottiva è indotta dalla iniezione intraperitoneale di GFP-etichettata Ova-specifiche CCR7 - effettrici / memoria linea cellulare T (Beeton, C J. Esperimenti Visualized, numero 8). Le cellule sono poi lasciato equilibrare nel ratto per 48 ore prima sfida, iniettando un orecchio con soluzione salina (controllo orecchio) e l&#39;altro con una miscela 1:1 di coniugati Ova e Ova a Texas-Red (Ova-TR) per consentire la visualizzazione residente di cellule presentanti l&#39;antigene. Descriviamo un metodo di preparazione dei tessuti utili per l&#39;imaging la motilità delle cellule all&#39;interno dello strato dermico profondo durante una risposta immunitaria, in collaborazione con visualizzazione delle fibre di collagene da generazione di seconda armonica. Il tessuto viene tagliato l&#39;orecchio in 5 x 5 millimetri quadrati (leggermente più grande è meglio), montato su slitta copertura in plastica con Vetbond ™, che sono poi protetti con grasso al silicone in una camera di imaging e superfused da ossigeno-bolle terreni di coltura a 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

Qui mostriamo un metodo per indurre la registrazione e il progresso di una ipersensibilità di tipo ritardato (DTH) reazione nell&#39;orecchio ratto. Questa è seguita da una dimostrazione della preparazione di tessuto di ratto orecchio per l&#39;imaging a due fotoni del effettrici / risposta delle cellule T memoria.

Imaging Effector cellule T di memoria nell&#39;orecchio dopo l&#39;induzione di DTH adottivi

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

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Biology

10.2K Views.  University of California, Irvine (UCI). Qui mostriamo un metodo per indurre la registrazione e il progresso di una ipersensibilità di tipo ritardato (DTH) reazione nell'orecchio ratto. Questa è seguita da una dimostrazione della preparazione di tessuto di ratto orecchio per l'imaging a due fotoni del effettrici / risposta delle cellule T memoria.

Video: 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.

Induzione e Monitoraggio di Active ipersensibilità di tipo ritardato (DTH) in ratti

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

Ricerca

Biologia

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.

Induzione e Monitoraggio adottivi di ipersensibilità ritardata nei ratti

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.

Clonogenica saggio: cellule aderenti

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.

Imaging del recettore dell&#39;estrogeno α in Rat Arteriole Pial utilizzando un microscopio digitale immunofluorescenza

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.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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.

Imaging locale Ca<sup&gt; 2+</sup&gt; Segnali in cellule di mammifero coltivate

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

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.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

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.

Recupero di fluorescenza dopo Photobleaching di giallo fluorescente proteine Tagged p62 in Aggresome-come le strutture indotta

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.

Dissezione e coltura efficienti delle cellule epiteliali del pigmento retino del topo primario

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