Induktion von Adoptive DTH Bitte beachten Sie JoVE Artikel: <a href="/index/Details.stp?ID=325">Induktion und Überwachung von Adoptive Allergien vom verzögerten Typ in Ratten</a> Christine Beeton, George K. Chandy Institut für Physiologie und Biophysik, University of California, Irvine J. Visualisierte Experimente, Issue 8 Hier haben wir dieses Protokoll mit Antigen (Ovalbumin), konjugiert mit Texas-Red in einem Verhältnis von 1:1 mit unmarkiertem Antigen um zur Visualisierung von Phagozyten Antigen-präsentierenden Zellen können modifiziert werden. Ohrgewebe Ernte <ol><li> Euthanize der Ratte in der gewünschten Zeit-Punkt durch Verfahren in Ihrem genehmigten Tier-Protokoll beschrieben. Hier verwenden wir eine Überdosis des Narkosemittels, Isofluran. Dies ist in einem geschlossenen Behälter in einem gut belüfteten Motorhaube getan. Stellen Sie sicher, das Tier tot ist entweder durch Enthauptung durch die Eröffnung der Brusthöhle und Schneiden des Herzens. </li><li> Mit großen Schere entfernen das Ohr mit einem einzigen Schnitt, möglichst nahe an den Körper des Tieres wie möglich (Abbildung 1). <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> Abbildung 1. Die Entfernung der Ratte Ohr </li><li> Legen Sie das Ohr in einer 15 ml konische Röhrchen mit ca. 10 ml eiskaltem 1x PBS oder RPMI-1640. Halten Sie die Gewebeproben auf Eis, bis Sie bereit, um das Gewebe für die Bildgebung herzustellen sind. </li><li> Bild des Gewebes so schnell wie möglich, wir finden aber, dass 1 bis 2 Stunden auf Eis keine erkennbare Wirkung auf die Zellbewegung in das Gewebe, wenn das Gewebe geerntet und abgebildet sofort verglichen. </li></ol> Tissue Vorbereitung für Imaging I. Entfernung von epidermalen und dermalen Schicht * Hinweis: Die tiefe Hautschicht muss beibehalten werden. Wenn es richtig gemacht großen Blutgefäße bleiben erhalten und der Knorpel des Ohres nicht ausgesetzt werden. Dies ist eine schwierige Technik, vor allem, wenn es keine Entzündung (Kontrollbedingungen) und es ist hilfreich, ein Binokular verwenden. <ol><li> Legen Sie beide das Gewebe und Medien aus dem konischen Rohr in einer 10 cm Gewebe Kulturschale. </li><li> Mit behandschuhten Händen halten die Ratte Ohr sanft an der Spitze des Ohres (distalen) mit der Dorsalseite des Ohres nach oben (Abbildung 2). <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> Abbildung 2. Trim Fell aus dem Ohr </li><li> Mit dem Daumen auf der Oberseite des Ohrs, steady das Gewebe während der Verwendung der Spitze des geschlossenen Dumont Nr. 5 Schere, um die Dermis aus der tiefen Dermis zu trennen. Alternativ, wenn der Rand des Ohres hat einen Überhang von Haut / tiefe Dermis, kann dies mit einer Pinzette fassen und vorsichtig herausgezogen, um diese aus dem Gewebe darunter (Abbildung 3) zu trennen. <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> Abbildung 3. Die Entfernung der oberen Dermis </li><li> Nach einem kleinen Bereich der Haut hat sich von dem darunter liegenden Gewebe abgetrennt wurde, wiederholen Sie die Schritte Trennung alternativ mit einer Schere zu schneiden zwischen den Hautschichten und Pinzette vorsichtig entfernen und Abziehen der Haut abgelöst. </li><li> Expose eine 5 x 5 mm oder größere Fläche Ohrgewebe, dass mindestens 3 mm von der ersten Seite der Antigen-Injektion (Abbildung 4). <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> Abbildung 4. Tissue für die Bildgebung ausgesetzt </li></ol> II. Sicherung Tissue Imaging zur Bühne <ol><li> Schneiden Sie ein Kunststoff Deckglas zu einer Größe etwas größer als vorbereitet Gewebe. </li><li> Verteilen Sie eine dünne Schicht von 3M Vetbond ™ Gewebe-safe Kleber auf den Objektträger. </li><li> Fassen Sie die Ecke der präparierten Gewebe (wo Sie sind nicht geeignet, Bild), von Medien zu entfernen, und tupfen Sie die Unterseite des Gewebes auf der Linse Papier oder Kym wischen, um überschüssige Medien zu entfernen. </li><li> Tippen Sie auf die Kante des Gewebes auf den Leim bedeckt Schlitten (die Vetbond härtet sofort auf nassen Gewebe). Vorsichtig dehnen das Gewebe flach, mit dem anderen Ende ist der letzte, der die Folie berühren (Abbildung 5) <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> Abbildung 5. Berg Gewebe Slip Cover </li><li> Cure den Vetbond ™ durch Umkehrung der Gewebeprobe / Rutsche und Eintauchen in ein Reservoir von Medien, so dass das Gewebe und ziehen nach unten und rund sind in einem Winkel von 45 Grad. Halten Sie das Gewebe auf den Kopf in einem Winkel hilft, überschüssigen Kleber aus Aushärtung an der exponierten Gewebeoberfläche verhindern. Kleber auf der Oberfläche des Gewebes blockiert den Laser (Abbildung 6). <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> Abbildung 6. Cure vetbond Gewebe glue durch Eintauchen in den Medien Hinweis: Die Schritte 2-5 können ein wenig Übung zu meistern. Versuchen Sie üben mit weggeworfenen oder extra Gewebestücke, bevor zum ersten Mal. </li><li> Dab eine kleine Menge Silikonfett auf den unteren Rand der Folie. </li><li> Legen Sie die Folie in der Perfusionskammer. Stellen Sie sicher, dass die Perfusion Medien ist fließend, 37 ° C und mit Sauerstoff versorgt, bevor Sie Gewebe. Drücken Sie vorsichtig auf die Ränder der Folie machen ein Siegel mit dem Silikonfett zwischen der Unterseite des Perfusionskammer und die Kunststoff-Deckglas. </li></ol> III. Zwei-Photonen-Imaging <ol><li> Mit Hellfeld-Durchlicht, auf der Oberseite des Gewebes zu konzentrieren. Schalten Sie alle Lichter. </li><li> Schalten Sie den Laser, PMTs wie von Ihrem Multi-Photonen-Mikroskop-System erforderlich. </li><li> Second Harmonic Generation (SHG) in hoch geordneten faserige Proteine ​​(Kollagen und Myosin) erzeugt ein Signal mit der halben Wellenlänge des zur Anregung verwendeten Lasers. Wir verwenden Anregung bei 900 nm, was zu SHG bei 450 nm, die visualisiert werden können mit dem &quot;blue&quot;-Kanal des Mikroskops. SHG-Generation ist eine nicht-lineare (Zwei-Photonen-) Prozess, und bietet somit eine optische &quot;Schneiden&quot;-Effekt ähnlich dem der Zwei-Photonen-Anregung der Fluoreszenz. Die maximale Effizienz des SHG tritt auf, wenn die Fibrillen senkrecht zur Laser, während Fibrillen, die parallel ausgeführt werden nicht visualisiert. 1 </li><li> Suchen Sie einen guten Imaging-Bereich mit reichlich Zellen, stellen Sie Ihren Erwerb Bereich, so dass die Mehrzahl der Zellen in der Mitte sind und beginnen Bildgebung. Hinweis: In DTH-Reaktionen, T-Zellen und anderen Zellen infiltriert sind oft zusammen konzentriert werden, während andere Bereiche frei von infiltrierenden Zellen 2 sind. Daher kann es einige Zeit dauern, die Suche im Gewebe um die beste Gegend, um Bild zu finden. </li><li> Prüfen Sie, ob die Lebensfähigkeit der Zellen durch die Beurteilung Zelle Geschwindigkeit und Polarität während der Bildgebung. Verwenden Sie die niedrigste Laserleistung, dass akzeptable Bildqualität bietet, um minimal Foto-Schäden zu gewährleisten. </li><li> Swap in eine neue Gewebe Vorbereitung wie nötig. </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

Verzögerte Überempfindlichkeitsreaktionen (DTH) ist eine Immun-Reaktion, bei der die wichtigsten Akteure sind CCR7 - Effektor / Memory T-Lymphozyten. Hier zeigen wir ein Verfahren zur Induktion und Aufzeichnung des Fortschritts einer DTH-Reaktion bei der Ratte Ohr. Effektor / Memory T-Zell-Antwort - Dies ist eine Demonstration der Zubereitung von Ratten Ohrgewebe für Zwei-Photonen-Imaging der CCR7 gefolgt. Effektor / Memory T-Zell-Linie (Beeton, C J. Visualisierte Experimente, Issue 8) - Ein Adoptiv-DTH wird durch die intraperitoneale Injektion von GFP-markierten Ova-spezifische CCR7 induziert. Die Zellen werden dann erlaubt, bei der Ratte für 48 Stunden ruhen, bevor Herausforderung durch die Injektion von einem Ohr mit Kochsalzlösung (Kontrollgruppe Ohr) und die andere mit einem 1:1-Mischung von Eizellen und Ova konjugiert Texas-Red (Ova-TR) zur Visualisierung ermöglichen der ansässigen Antigen-präsentierenden Zellen. Wir beschreiben eine Methode, Gewebe Vorbereitung nützlich für die Bildgebung die Motilität von Zellen in den tiefen Hautschichten während einer Immunantwort in Verbindung mit Visualisierung von Kollagenfasern durch Erzeugung der zweiten Harmonischen. Ear Gewebe in 5 x 5 mm Quadrate (etwas größer ist besser) geschnitten und auf Kunststoff Deckgläser mit Vetbond ™, die dann gesichert werden mit Silikonfett in einem bildgebenden Kammer und superfundiert durch Sauerstoff-Blasen Gewebekultur-Medium bei 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

Hier zeigen wir ein Verfahren zur Induktion und Aufzeichnung des Fortschritts einer verzögerten Typ-Überempfindlichkeit (DTH)-Reaktion bei der Ratte Ohr. Dies wird durch eine Demonstration der Vorbereitung der Ratte Ohrgewebe für Zwei-Photonen-Imaging der Effektor / Memory T-Zell-Antwort folgte.

Imaging Effektor-Memory-T-Zellen im Ohr nach Induktion der Adoptive DTH

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

Research

JoVE Journal

Biology

10.2K Aufrufe.  University of California, Irvine (UCI). Hier zeigen wir ein Verfahren zur Induktion und Aufzeichnung des Fortschritts einer verzögerten Typ-Überempfindlichkeit (DTH)-Reaktion bei der Ratte Ohr. Dies wird durch eine Demonstration der Vorbereitung der Ratte Ohrgewebe für Zwei-Photonen-Imaging der Effektor / Memory T-Zell-Antwort folgte.

Serie: 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.

Induktion und Überwachung von Active Delayed Type Überempfindlichkeit (DTH) in Ratten

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

Forschung

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.

Induktion und Überwachung von Adoptive Allergien vom verzögerten Typ in Ratten

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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.

Klonogene Test: adhärenter Zellen

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

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Live-Bildgebung von Zell Extrusion aus der Epidermis der Entwicklung von Zebrafisch

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

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-&#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 von Estrogen Receptor-α in Rat Pial Arteriolen mit einem Digital-Immunfluoreszenz-Mikroskop

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluoreszenz-Imaging mit One-Nanometer-Genauigkeit (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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 Lokale Ca<sup&gt; 2+</sup&gt; Signale in kultivierten Säugetierzellen

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.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

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