1. Preparazione della emulsione <ol><li> L&#39;emulsione è una miscela 1:1 di antigene (DTH può essere indotto a vari antigeni: ovoalbumina, tossoide tetanico, buco della serratura patelle hemocyanin ....) in soluzione acquosa aggiunto per completare adiuvante di Freund. E &#39;molto importante aggiungere l&#39;antigene per l&#39;adiuvante e non viceversa! </li><li> Ci sono un sacco di rifiuti al momento e iniettando una emulsione, in modo sempre preparare 1,5 o 2 volte più di quello che vi serve. </li><li> Sciogliere l&#39;antigene in soluzione fisiologica per preparare un 2 mg / ml soluzione. Non filtrare, come non si vuole perdere la aggregati! </li><li> Mettete adiuvante completo di Freund H37Ra (Difco, catalogo # 231131) in una provetta e miscelare ad alta velocità. Aggiungere la goccia a goccia, mentre l&#39;antigene vortex. Quando tutti l&#39;antigene è aggiunto, vortex per 5 minuti. La miscela dovrebbe diventare bianco. </li><li> Mettere l&#39;emulsione in una siringa di vetro e collegarlo ad un altro siringa in vetro con un ponte 18G (catalogo Fisher # 14-825-17L). Invia l&#39;emulsione da una siringa all&#39;altra fino a quando diventa difficile (5-10 minuti). E &#39;meglio utilizzare due siringhe da 5 ml. Se più di 5 ml di emulsione sono necessari, usano vari gruppi di 5 siringhe ml. </li><li> L&#39;emulsione può essere memorizzato in siringhe a 4 ° C per 3 settimane. </li></ol> 2. Immunizzazione dei topi <ol><li> Mescolare l&#39;emulsione nelle siringhe per qualche minuto e il trasferimento alla siringa usata per l&#39;iniezione. Iniettare 200 ml per via sottocutanea in 1-2 siti alla base della coda con aghi 23G e una Luer-Lok tip siringa da 3 ml. </li></ol> 3. Sfida con l&#39;antigene nella parte posteriore <ol><li> Sette giorni dopo l&#39;immunizzazione, barba 2 spot sul dorso dei topi e intradermica iniettare 20 ml di una mg / ml soluzione 1 antigene in un punto e soluzione salina (o antigene irrilevante) nel punto altri. Non filtrare la soluzione antigene, come si può perdere aggregati che aumentare la reazione infiammatoria. </li><li> I ratti devono essere anesthesized per evitare movimenti. Iniettare la soluzione intradermica con 27G ½ aghi e siringhe da 1 ml. Cambia aghi tra le iniezioni. Nota: la sfida può essere fatto nel padiglione delle orecchie al posto della schiena per misure più precise. Guarda il video a <a href="/index/Details.stp?ID=325">https://www.jove.com/index/Details.stp?ID=325</a> </li></ol> 4. Misura della DTH <ol><li> Il sito di iniezione inizierà a girare rosso e gonfiore in segno di infiammazione. Misurare il diametro della zona infiammata con un righello o, meglio ancora, una pinza. Il gonfiore massimo dovrebbe avvenire tra il 24 e 72 ore. </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>

Gli autori sono co-fondatori e consulenti per Airmid Inc.

Per una misurazione più precisa della reazione DTH si consiglia sfida i topi con l&#39;antigene nel padiglione dell&#39;orecchio. La reazione può essere misurata come differenza all&#39;orecchio gonfiore tra le pertinenti antigene-sfidato l&#39;orecchio e l&#39;orecchio sfidato sia con soluzione salina da solo o con l&#39;antigene irrilevante. Una dimostrazione della sfida a livello dell&#39;orecchio mostrato in 1.

Utilizzare i reagenti sterili soluzioni e siringhe. Le siringhe di vetro e ponti 18G possono essere sterilizzati in autoclave. Leggi materiale scheda prima di maneggiare un reagente per la prima volta e indossare equipaggiamento protettivo.

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

Ipersensibilità di tipo ritardato (DTH) è una reazione infiammatoria mediata dai linfociti memoria CCR7-T effettrici che si infiltrano nel sito di iniezione di un antigene contro il quale il sistema immunitario è stato innescato. La reazione infiammatoria è caratterizzata da arrossamento e gonfiore nel sito di uno stimolo antigenico. Si tratta di un modello pratico per determinare l&#39;efficacia in vivo di immunosoppressori. DTH cutanea può essere indotta in seguito a trasferimenti adottivo di antigene-specifica dei linfociti T o per l&#39;immunizzazione attiva con un antigene, e la successiva sfida intradermica con l&#39;antigene per indurre la reazione infiammatoria della pelle in una zona determinata. Risposte DTH può essere indotto a vari antigeni, per esempio ovalbumina, tubercolina, tossoide tetanico, o buco della serratura patelle hemocyanin (KLH). Qui mostriamo come per indurre una reazione attiva DTH nei ratti Lewis. Per prima cosa preparare un acqua in emulsione di olio KLH, antigene di nostro interesse, in adiuvante completo di Freund e questa emulsione iniettare per via sottocutanea nei ratti. Questo sarà il primo sistema immunitario di sviluppare cellule T di memoria diretta a KLH. Sette giorni dopo ci sarà una sfida per i ratti intradermica sul retro con KLH da un lato e con ovalbumina, un antigene irrilevante, sul lato opposto. La reazione infiammatoria sarà visibile 16-72 ore più tardi e la zona rossa e gonfia sarà misurata come indice di gravità DTH.

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

Ipersensibilità di tipo ritardato (DTH) è una reazione infiammatoria mediata dai linfociti memoria CCR7-T effettrici. Qui mostriamo come per indurre DTH attivo nei ratti Lewis e monitorare la risposta infiammatoria.

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 ...

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

Research

JoVE Journal

Biology

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

17.6K Views.  University of California, Irvine (UCI). Ipersensibilità di tipo ritardato (DTH) è una reazione infiammatoria mediata dai linfociti memoria CCR7-T effettrici. Qui mostriamo come per indurre DTH attivo nei ratti Lewis e monitorare la risposta infiammatoria.

Video: 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.

Induzione e Monitoraggio adottivi di ipersensibilità ritardata nei ratti

Ricerca

Biologia

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 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 ...

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.

Test di induzione neurale nell&#39;embrione Chick

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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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.

Induzione e test di ipossia in coltura cellulare

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.

Purificazione cromatografica dei ribosomi di lievito Highly Active

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.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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.

A lungo termine del test di tossicità letale con il crostaceo 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 Sintesi di mRNA modificata per l&#39;induzione di espressione della proteina nelle cellule umane

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.