1. Herstellung der Emulsion <ol><li> Die Emulsion ist eine 1:1-Mischung von Antigen (DTH kann auf verschiedene Antigene induziert werden: Ovalbumin, fügte Tetanustoxoid, Napfschnecken-Hämocyanin ....) in wässriger Lösung, um Freund-Adjuvans abzuschließen. Es ist sehr wichtig, um das Antigen an das Adjuvans und nicht umgekehrt hinzuzufügen! </li><li> Es gibt eine Menge von Abfällen, wenn Herstellung und Injektion einer Emulsion, so dass immer vorbereiten 1,5 oder 2-mal mehr als das, was Sie brauchen. </li><li> Lösen Sie Antigen in Kochsalzlösung zu einer 2 mg / ml Lösung herzustellen. Nicht filtern, weil Sie nicht möchten, dass die Aggregate locker! </li><li> Put komplettem Freund-Adjuvans H37Ra (Difco, Katalog # 231131) in einer Röhre und Wirbel mit hoher Geschwindigkeit. Fügen Sie die Antigen tropfenweise unter Vortexen. Wenn alle das Antigen zugesetzt wird, Wirbel für weitere 5 Minuten. Die Mischung sollte sich weiß. </li><li> Legen Sie die Emulsion in eine Glasspritze und verknüpfen es mit einem anderen Glasspritze mit einer 18G-Brücke (Fisher Katalog # 14-825-17L). Senden Sie die Emulsion aus einer Spritze auf der anderen, bis sie hart (5-10 min) wird. Am besten ist es, zwei 5 ml Spritzen verwenden. Wenn mehr als 5 ml Emulsion benötigt werden, verwenden Sie mehrere Sätze von 5 ml Spritzen. </li><li> Die Emulsion kann in die Spritzen bei 4 ° C für 3 Wochen gelagert. </li></ol> 2. Die Immunisierung der Ratten <ol><li> Mix der Emulsion in die Spritzen für ein paar Minuten und Transfer zum Spritzen für die Injektion verwendet. Inject 200 ml subkutan in 1-2 Seiten an der Basis des Schwanzes mit 23G Nadeln und ein Luer-Lok Spitze 3 ml Spritze. </li></ol> 3. Challenge mit Antigen in den Rücken <ol><li> Sieben Tage nach der Immunisierung, rasieren 2 Spots auf dem Rücken der Ratten und injizieren intradermal 20 ml einer 1 mg / ml Antigen-Lösung auf einem Fleck und Kochsalzlösung (oder irrelevant Antigen) in den anderen Ort. Nicht filtern die Antigen-Lösung, wie man Aggregate, die die Entzündungsreaktion verstärken können locker. </li><li> Die Ratten sollten narkotisiert, um Bewegungen zu vermeiden. Injizieren Sie die Lösung intradermal mit 27G ½ Nadeln und 1 ml Spritzen. Ändern Nadeln zwischen den Injektionen. Hinweis: Die Herausforderung in der Ohrmuschel der Ohren statt an der Rückseite für genauere Messungen durchgeführt werden. Siehe Video bei <a href="/index/Details.stp?ID=325">https://www.jove.com/index/Details.stp?ID=325</a> </li></ol> 4. Messung von DTH <ol><li> Die Einstichstelle beginnt Rötung und Schwellung als Zeichen der Entzündung. Messen Sie den Durchmesser des entzündeten Bereich mit einem Lineal oder noch besser, haben eine Dicke. Die maximale Schwellung sollte zwischen 24 und 72 Stunden auftreten. </li></ol>

<ol>
	<li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+monitoring+of+adoptive+delayed-type+hypersensitivity+in+rats.">Induction and monitoring of adoptive delayed-type hypersensitivity in rats.</a> Journal of Visualized Experiments. 8, (2007).</li></ol>

Die Autoren sind Mitbegründer und Berater für Airmid Inc.

Für eine genauere Messung der DTH-Reaktion empfehlen wir gegen die Ratten, die mit dem Antigen in der Ohrmuschel. Die Reaktion kann dann als Differenz in Ohrschwellung zwischen den relevanten Antigen-herausgefordert Ohr und das Ohr entweder mit Kochsalzlösung alleine oder mit dem irrelevanten Antigen herausgefordert gemessen werden. Eine Demonstration der Herausforderung im Ohr ist in 1 dargestellt.

Verwenden Sie sterile Lösungen Reagenzien und Spritzen. Die Glas-Spritzen und 18G Brücken können autoklaviert werden. Lesen Sie Material Datenblatt vor dem Umgang mit einem Reagenz zum ersten Mal und angemessene Schutzkleidung.

induction and monitoring of active delayed type hypersensitivity (dth) in rats

Verzögerte Überempfindlichkeitsreaktionen (DTH) ist eine entzündliche Reaktion durch CCR7-Effektor-Memory-T-Lymphozyten, dass der Injektionsstelle eines Antigens, gegen die das Immunsystem grundiert hat infiltrieren vermittelt. Die Entzündungsreaktion wird durch Rötung und Schwellung der Website von antigenen Herausforderung geprägt. Es ist eine bequeme Modell, um die in vivo Wirksamkeit von Immunsuppressiva zu bestimmen. Kutane DTH kann entweder durch adoptiven Transfer von Antigen-spezifischen T-Lymphozyten oder durch aktive Immunisierung mit einem Antigen, und die anschließende intradermal Herausforderung mit dem Antigen auf die entzündliche Reaktion in einem bestimmten Hautareal hervorrufen induziert werden. DTH-Reaktionen können auf verschiedene Antigene hervorgerufen werden, z. B. Ovalbumin, Tuberkulin-, Tetanus-Toxoid oder Napfschnecken-Hämocyanin (KLH). Hier zeigen wir, wie eine aktive DTH-Reaktion in Lewis-Ratten zu induzieren. Wir werden zunächst ein Wasser-in-Öl-Emulsion von KLH, unser Antigen von Interesse, in komplettem Freund-Adjuvans und injizieren diese Emulsion subkutan bei Ratten. Dies wird prime das Immunsystem zu Gedächtnis-T-Zellen gerichtet an KLH zu entwickeln. Sieben Tage später werden wir die Ratten intradermal Herausforderung auf der Rückseite mit KLH auf der einen Seite und mit Ovalbumin, ein irrelevantes Antigen, auf der anderen Seite. Die entzündliche Reaktion sichtbar sein wird 16-72 Stunden später und die rot und geschwollen Bereich wird als Indiz für DTH Schwere gemessen werden.

Watch this Scientific Journal Video about Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats at JoVE.com

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Verzögerte Überempfindlichkeitsreaktionen (DTH) ist eine entzündliche Reaktion durch CCR7-Effektor-Memory-T-Lymphozyten vermittelt. Hier zeigen wir, wie die aktive DTH in Lewis-Ratten induzieren und Überwachung der entzündlichen Reaktion.

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 ...

induction-monitoring-active-delayed-type-hypersensitivity-dth

Research

JoVE Journal

Biology

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

17.7K Aufrufe.  University of California, Irvine (UCI). Verzögerte Überempfindlichkeitsreaktionen (DTH) ist eine entzündliche Reaktion durch CCR7-Effektor-Memory-T-Lymphozyten vermittelt. Hier zeigen wir, wie die aktive DTH in Lewis-Ratten induzieren und Überwachung der entzündlichen Reaktion.

Serie: Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

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

Forschung

Biologie

Imaging Effector Memory T cells in the Ear After Induction of 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 method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7- effector / memory T cell response.
An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.
We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond&trade;, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37&deg;C.

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

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 ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Assay für Neural Induction im Hühnerembryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Rekonstitution der Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induktion und Prüfung von Hypoxie in der Zellkultur

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Chromatographische Reinigung von hoch aktiven Hefe Ribosomen

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Langfristige letale Toxizität Test mit dem Krustentier Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

In vitro Synthese von modifizierten mRNA zur Induktion der Proteinexpression in menschlichen Zellen

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.