Agradecemos ao Dr. Alexander Flugel (Martinsried, Alemanha) para fornecer-nos com a GFP-rotulados, linha de células T específicas ovalbumina utilizado neste protocolo.

1. Estimulação dos linfócitos T específicos ovalbumina Descansando células ovalbumina específico T são estimuladas com ovalbumina na presença de ratos irradiados timócitos Lewis (30 Gy) como células apresentadoras de antígenos (APCs), no Médio Estimulação (Prepare em DMEM + Pen / Strep / L-Glut + + NEAA RPMI Vitaminas + + 2ME Rat Serum + ovalbumina, nas concentrações finais observou na lista de materiais.) Preparação do soro de ratos <ol><li> Tomamos soro dos ratos nos sacrificamos como doadores timo. </li><li> Os ratos são profundamente anestesiado por inalação de halotano e tanto sangue quanto possível é desenhada por perfusão cardíaca (50-10 ml por rato), utilizando 10 seringas ml e 23 G 1 &quot;agulhas, após o que os ratos são imediatamente decapitado para garantir a morte. </li><li> O sangue é permitida a coagular por 10 min a 37 ° C seguido por 10 min no gelo. </li><li> O coágulo é removido eo líquido restante fiadas. </li><li> O soro é coletado, filtrado estéril e armazenado a -20 ° C. </li></ol> Preparação das células apresentadoras de antígenos <ol><li> Leve o timo de um rato decapitado utilizando instrumentos estéreis e colocá-lo em PBS contendo penicilina e estreptomicina (PBS-PS), sobre o gelo. </li><li> Leve o timo de um capuz de cultura de tecidos, limpe-o de coágulos sanguíneos e tecido contaminar outras e corte-o em pequenos pedaços em uma peneira de células (Fisher # 08-771-2) colocado em um prato de 10 centímetros de petri contendo 10 ml de PBS- PS. </li><li> Cada peça é pressionado através do filtro de células usando as costas de um êmbolo da seringa estéril 1 ml. Recolher a suspensão única célula em um tubo de 50 ml no gelo. </li><li> Lavar o filtro de células com 20-40 ml de PBS mais-PS. Centrifugar a suspensão de 1250g e ressuspender o sedimento em PBS-PS, 5 ml / timo, sobre o gelo. </li><li> Irradiar esta suspensão (30 Gy) em um irradiador gama e lave-o duas vezes com 50 ml de PBS-PS. Nota: O melhor doadores timo são ratos adultos jovens, 5-7 semanas de idade. Ratos até 10 semanas pode ser usado, mas o número de células diminui com o tempo. Sexo não é importante, embora algumas pessoas dizem que os homens são melhores doadores timo. Nós sempre usamos fêmeas porque todos os nossos animais são alojados no mesmo quarto e nós não queremos homens e mulheres na mesma sala. </li></ol> Estimulação <ol><li> Lavar os linfócitos T uma vez e contá-las. </li><li> Contar as células apresentadoras de antígenos. </li><li> Mix 3 x 10 6 ovalbumina específicas de células T com 150 x 10 6 APCs em um prato de 10 centímetros de petri contendo 10 ml de meio de estimulação. </li><li> Incubar por 48 horas a 37 ° C. </li></ol> 2. Transferência adotiva dos linfócitos T específicos ovalbumina <ol><li> Os ratos que usamos como destinatários são do sexo feminino 70-10 semanas de idade. </li><li> Contar os linfócitos T, elas devem ser grandes e redondos. As células apresentadoras de antígenos será morto. </li><li> Centrifugar os linfócitos T e ressuspenda-los em 10 x 10 6 células / ml em PBS. </li><li> Injectar 1 ml por via intraperitoneal com 3 seringas ml e 23 G 1 &quot;agulhas. </li></ol> 3. Desafio com antígeno no ouvido <ol><li> Dois dias após a transferência adotiva, injetar 20 l de 1 mg / ml solução ovalbumina (= antígeno relevante) por um ouvido e salinas (ou antígeno irrelevante) na outra orelha. Não filtrar a solução antígeno como você pode perder agregados que aumentam a reação inflamatória. Nota: Os ratos devem ser anestesiados para evitar movimentos. Se você é destro, segure a orelha entre o polegar eo dedo indicador esquerdo, o dedo indicador estar dentro da orelha e do rato voltado para você. </li><li> Injetar a solução no pavilhão da orelha usando 27G ½ &quot;agulhas. Tente injetar todos os ratos exatamente no mesmo lugar. </li></ol> 4. Medição de DTH <ol><li> O ouvido começa a ficar vermelha e inchar como um sinal de inflamação. Medir a espessura de cada orelha com um micrômetro de mola (por exemplo modelo PK-0505 da Mitutoyo). </li><li> Tome 5-6 medições para cada orelha em cada ponto do tempo e calcular a média. </li><li> O inchaço máxima deve ocorrer entre 24 e 72 horas. </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>

O desafio antigênico também pode ser realizada em outros locais, por exemplo, na pele do dorso (2). Nós, no entanto descobrir que um desafio. Usando o pavilhão da orelha, como mostra este vídeo, permite a medição mais precisa da reação DTH.

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco/Invitrogen</td>
<td>12430-062</td>
<td>500ml</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>1 mM final, in Stimulation medium</td>
</tr>
<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>
</tr>
<tr>
<td>RPMI vitamins</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7256</td>
<td>1% final, in Stimulation medium</td>
</tr>
<tr>
<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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<tr>
<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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<tr>
<td>Ovalbumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5503</td>
<td>10 &micro;g/ml final, in Stimulation Medium.</td>
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induction and monitoring of adoptive delayed-type hypersensitivity in rats

De hipersensibilidade do tipo retardado (DTH) é uma reação inflamatória mediada por linfócitos efetores CCR7-T de memória que infiltram o local da injeção de um antígeno contra o qual o sistema imunológico tem sido preparado. A reação inflamatória é caracterizada por vermelhidão e inchaço do local de desafio antigênico. É um modelo conveniente para determinar a eficácia in vivo de imunossupressores. DTH cutânea pode ser induzida ou por transferência adotiva de linfócitos T antígeno-específicos ou por imunização ativa com um antígeno e subseqüente desafio intradérmico com o antígeno para induzir a reação inflamatória da pele em uma área determinada. Respostas DTH pode ser induzida a vários antígenos, para a ovalbumina exemplo, a tuberculina, toxóide tetânico, ou hemocianina limpet buraco da fechadura. Tais reações também pode ser induzida contra autoantígeno, por exemplo, para proteína básica da mielina (MBP) em ratos com encefalomielite auto-imune experimental induzida com MBP, um modelo animal para a esclerose múltipla (1). Aqui demonstramos como para induzir uma reação adotivos DTH em ratos Lewis. Vamos primeiro estimulam as células T específicas ovalbumina in vitro e injetar essas células ativadas por via intraperitoneal em ratos ingênuos. Depois de permitir que as células para equilibrar in vivo por 2 dias, vamos desafiar os ratos com ovalbumina no pavilhão auricular de uma orelha, enquanto a outra orelha wil receber solução salina. A reação inflamatória será visível 3-72 horas depois e espessura de ouvido será medido como uma indicação da gravidade da DTH.

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

De hipersensibilidade do tipo retardado (DTH) é uma reação inflamatória mediada por CCR7-efetoras de memória T (TEM) linfócitos. Aqui demonstramos como ativar células antígeno-específicas TEM, induzir DTH adotivos em ratos Lewis e monitorar a resposta inflamatória.

Indução e Monitoramento de hipersensibilidade tardia-Type Adoptive em Ratos

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 visualizações.  University of California, Irvine (UCI). De hipersensibilidade do tipo retardado (DTH) é uma reação inflamatória mediada por CCR7-efetoras de memória T (TEM) linfócitos. Aqui demonstramos como ativar células antígeno-específicas TEM, induzir DTH adotivos em ratos Lewis e monitorar a resposta inflamatória.

Vídeo: 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.

Indução e Monitoramento de hipersensibilidade do tipo retardada Ativa (DTH) em ratos

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.

As células de memória efetoras T imagem no ouvido após a indução de DTH Adoptive

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.

Ensaio para Indução Neural no embrião de galinha

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.

Alterações na morfologia da glândula mamária e risco de câncer de mama em ratos

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.

Indução de hipóxia e Testes em Cultura de Células

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.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

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 longo prazo de Teste de toxicidade letal com o crustáceo 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.

Indução e Análise de epitelial para mesenquimal Transição

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.

In Vitro Síntese de ARNm de modificação para indução da expressão da proteína em células humanas

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.