Nous remercions le Dr Alexander Flügel (Martinsried, Allemagne) pour nous avoir fourni la GFP-étiquetée, l&#39;ovalbumine spécifiques lignée cellulaire T utilisé dans ce protocole.

1. La stimulation des lymphocytes T spécifiques d&#39;ovalbumine Repos ovalbumine cellules T spécifiques sont stimulés avec de l&#39;ovalbumine en présence des thymocytes irradiés de rats Lewis (30 Gy) comme des cellules présentatrices d&#39;antigènes (APC), en milieu de stimulation (Préparer dans du DMEM + Pen / Strep / L-Glut + + NEAA RPMI Vitamines + + 2ME sérum de rat + ovalbumine, à la concentration finale noté dans la liste des matériaux.) Préparation de sérum de rat <ol><li> Nous prenons sérum du rat nous sacrifier en tant que donateurs thymus. </li><li> Les rats sont profondément anesthésiés par inhalation d&#39;halothane et autant de sang que possible est dessiné par perfusion cardiaque (5-10 ml par rat) en utilisant 10 ml et les seringues 23 G 1 &quot;aiguilles, après laquelle les rats sont immédiatement décapités pour assurer la mort. </li><li> Le sang est autorisé à se coaguler pendant 10 min à 37 ° C suivi de 10 min sur la glace. </li><li> Le caillot est retiré et le liquide restant filé. </li><li> Le sérum est recueilli, stérilisée par filtration et conservés à -20 ° C. </li></ol> Préparation des cellules présentatrices d&#39;antigènes <ol><li> Prenez le thymus d&#39;un rat décapité à l&#39;aide d&#39;instruments stériles et placez-le dans du PBS contenant de la pénicilline et la streptomycine (PBS-PS), sur la glace. </li><li> Prenez le thymus pour une hotte de culture de tissus, de le nettoyer de caillots de sang et autres tissus contaminant et le couper en petits morceaux dans une passoire cellule (Fisher # 08-771-2) placé dans une boîte de Pétri de 10 cm contenant 10 ml de PBS- PS. </li><li> Chaque pièce est pressé à travers le tamis de cellules en utilisant le dos d&#39;un piston de la seringue stérile de 1 ml. Recueillir la suspension seule cellule dans un tube de 50 ml sur la glace. </li><li> Rincer la crépine cellule avec plus de 20-40 ml de PBS-PS. Centrifuger la suspension à 1250g et resuspendre le culot dans du PBS-PS, 5 ml / thymus, sur la glace. </li><li> Irradier cette suspension (30 Gy) dans un irradiateur gamma et immédiatement laver deux fois avec 50 ml de PBS-PS. Remarque: Le meilleur des donateurs du thymus sont de jeunes rats adultes, des semaines 5-7 ans. Les rats jusqu&#39;à 10 semaines peuvent être utilisés mais le nombre de cellules diminue avec le temps. Sexe n&#39;est pas important, bien que certaines personnes disent que les hommes sont mieux donateurs thymus. Nous utilisons toujours les femmes parce que tous nos animaux sont logés dans la même chambre et nous ne voulons pas les mâles et les femelles dans la même pièce. </li></ol> La stimulation <ol><li> Lavez les lymphocytes T une fois et les compter. </li><li> Compter les cellules présentatrices d&#39;antigène. </li><li> Mélanger 3 x 10 6 ovalbumine cellules T spécifiques de 150 x 10 6 TTB dans un plat de 10 cm de Pétri contenant 10 ml de milieu de stimulation. </li><li> Incuber pendant 48 heures à 37 ° C. </li></ol> 2. Le transfert adoptif de lymphocytes T spécifiques d&#39;ovalbumine <ol><li> Les rats que nous utilisons en tant que bénéficiaires sont des femmes de 70 à 10 semaines. </li><li> Comptez le nombre de lymphocytes T, ils doivent être grands et ronds. Les cellules présentatrices d&#39;antigène sera mort. </li><li> Centrifuger les lymphocytes T et les remettre en suspension à 10 x 10 6 cellules / ml dans le PBS. </li><li> Injecter 1 ml par voie intrapéritonéale en utilisant 3 ml et les seringues 23 G 1 &quot;aiguilles. </li></ol> 3. Défi avec l&#39;antigène dans l&#39;oreille <ol><li> Deux jours après le transfert adoptif, injecter 20 ul d&#39;une solution à 1 mg / ml d&#39;ovalbumine (antigène = lieu) dans une oreille et une solution saline (ou antigène non pertinent) dans l&#39;autre oreille. Ne pas filtrer la solution d&#39;antigène que vous risquez de perdre des agrégats qui améliorent la réaction inflammatoire. Remarque: Les rats doivent être anesthésiés pour éviter les mouvements. Si vous êtes droitier, tenez l&#39;oreille entre le pouce et l&#39;index gauche, l&#39;index étant à l&#39;intérieur de l&#39;oreille et le rat en face de vous. </li><li> Injectez la solution dans le pavillon de l&#39;oreille à l&#39;aide 27G ½ &quot;aiguilles. Essayez d&#39;injecter tous les rats exactement au même endroit. </li></ol> 4. Mesure de la SRD <ol><li> Les oreilles vont commencer à tourner rouge et gonflent en tant que signe d&#39;inflammation. Mesurer l&#39;épaisseur de chaque oreille avec un micromètre à ressort (pour le modèle par exemple PK-0505 de Mitutoyo). </li><li> Prendre 5-6 mesures pour chaque oreille à chaque point dans le temps et calculer la moyenne. </li><li> Le gonflement maximal devrait se produire entre 24 et 72 heures. </li></ol>

<ol>
	<li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> J. Immunol. , 166-936 (2001).</li><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+active+delayed+type+hypersensitivity+(DTH)in+rats.">Induction and monitoring of active delayed type hypersensitivity (DTH)in rats.</a> J Vis Exp. (5), (2007).</li></ol>

La stimulation antigénique peut également être effectuée à d&#39;autres sites, par exemple dans la peau du dos (2). Nous avons cependant trouvé que la contestation. Utilisation du pavillon de l&#39;oreille, comme le montre cette vidéo, permet la mesure la plus précise de la réaction de DTH.

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		<th>Type</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco/Invitrogen</td>
<td>12430-062</td>
<td>500ml</td>
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<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>1 mM final, in Stimulation medium</td>
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<tr>
<td>Penicillin &#x2013; Streptomycin &#x2013; L-Glutamine</td>
<td>&nbsp;</td>
<td>Cambrex/BioWhittaker</td>
<td>17-718R</td>
<td>100 u/ml - 100 &micro;g/ml - 4 mM respectively, final, in Stimulation Medium </td>
</tr>
<tr>
<td>Non essential amino acids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7131</td>
<td>NEAA, 1% final, in Stimulation Medium</td>
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<td>RPMI vitamins</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7256</td>
<td>1% final, in Stimulation medium</td>
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<td>beta-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6250</td>
<td>2ME, 50 &micro;M final, in Stimulation Medium</td>
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<td>Syngeneic rat serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1% final. See explanation in Protocol.</td>
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<td>Ovalbumin</td>
<td>&nbsp;</td>
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<td>A5503</td>
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induction and monitoring of adoptive delayed-type hypersensitivity in rats

Type de l&#39;hypersensibilité retardée (DTH) est une réaction inflammatoire médiée par CCR7-lymphocytes T effecteurs mémoire qui infiltrent le site de l&#39;injection d&#39;un antigène contre lequel le système immunitaire a été amorcée. La réaction inflammatoire est caractérisée par une rougeur et une enflure du site de stimulation antigénique. C&#39;est un modèle pratique pour déterminer l&#39;efficacité in vivo d&#39;immunosuppresseurs. SRD cutanée peut être induite soit par transfert adoptif de lymphocytes T spécifiques d&#39;antigènes ou par l&#39;immunisation active avec un antigène, et les suivantes défi intradermique avec l&#39;antigène pour induire la réaction inflammatoire dans une zone de peau donnés. Réponses DTH peuvent être induites à des antigènes différents, par exemple pour l&#39;ovalbumine, la tuberculine, anatoxine tétanique, ou hémocyanine de patelle. Ces réactions peuvent aussi être induite contre les autoantigènes, par exemple pour la protéine de myéline de base (MBP) chez le rat avec l&#39;encéphalomyélite auto-immune expérimentale induite par le MBP, un modèle animal de sclérose en plaques (1). Ici nous démontrons comment induire une réaction adoptifs SRD chez le rat Lewis. Nous allons d&#39;abord stimuler les cellules T spécifiques de l&#39;ovalbumine, in vitro et injecter ces cellules activées par voie intrapéritonéale à des rats naïfs. Après avoir laissé les cellules in vivo pour s&#39;équilibrer pendant 2 jours, nous mettra au défi les rats avec de l&#39;ovalbumine dans le pavillon d&#39;une oreille, tandis que l&#39;autre oreille wil recevez saline. La réaction inflammatoire sera visible 3-72 heures plus tard et l&#39;épaisseur de l&#39;oreille va être mesurée comme une indication de la gravité de SRD.

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

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Type de l&#39;hypersensibilité retardée (DTH) est une réaction inflammatoire médiée par CCR7-effectrices T mémoire (TEM) des lymphocytes. Ici nous démontrons comment activer les cellules spécifiques de l&#39;antigène TEM, induisent SRD adoptifs chez le rat Lewis et de surveiller la réponse inflammatoire.

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

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

induction-monitoring-adoptive-delayed-type-hypersensitivity

Research

JoVE Journal

Biology

11.8K vues.  University of California, Irvine (UCI). Type de l'hypersensibilité retardée (DTH) est une réaction inflammatoire médiée par CCR7-effectrices T mémoire (TEM) des lymphocytes. Ici nous démontrons comment activer les cellules spécifiques de l'antigène TEM, induisent SRD adoptifs chez le rat Lewis et de surveiller la réponse inflammatoire.

Vidéo: Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

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

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

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

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

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

Recherche

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.

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

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

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.

Dosage de l&#39;induction neurale dans l&#39;embryon de poulet

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

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Changements dans la morphologie glande mammaire et le risque de cancer du sein chez les rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

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.

Induction et les essais de l&#39;hypoxie en culture cellulaire

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 ...

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.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

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.

À long terme des essais de toxicité létale avec le Crustacé Artemia franciscana</em

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

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

Induction et analyse des épithéliale à mésenchymateuses transition

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

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.

La synthèse in vitro de l&#39;ARNm modifié pour l&#39;induction de l&#39;expression des protéines dans les cellules humaines

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

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

Flux de travail protéomique quantitative utilisant la détection basée sur la surveillance des réactions multiples des protéines du tissu cérébral humain