우리는이 프로토콜에서 사용되는 GFP - 라벨, ovalbumin 특정 T 세포 라인으로 우리를 제공할 박사 알렉산더 Flugel을 (Martinsried, 독일) 감사합니다.

1. ovalbumin 특정 T의 lymphocytes의 자극 휴식 ovalbumin 특정 T 세포는 자극을 매체 항원 제시 세포 (APCs)로 조사 루이스 랫 thymocytes (30 쥐), (DMEM + 펜 / Strep / L - GLUT + NEAA + RPMI의 비타민 준비의 존재에 ovalbumin로 자극된 상태네 + 2ME + 랫 세럼 + Ovalbumin, 최종 농도에서는 자료 목록에 언급했다.) 쥐 혈청 준비 <ol><li> 우리는 thymus 기증자로 희생 쥐의 혈청을. </li><li> 쥐들이 할로탄 흡입에 의해 깊이 anesthetized하고 가능한 한 많은 혈액으로 10 ML 주사기와 쥐 즉시 죽음을 보장하기 위해 목이있는 후 23 G 1 &quot;바늘을 사용하여 심장 재관류 (쥐 당 50-10 ML)에 의해 그려진 것입니다. </li><li> 혈액은 37 10 분 응고 수 있습니다 ° C 얼음이 10 분 뒤에. </li><li> 응고를 제거하고 남은 액체 돌다가 있습니다. </li><li> 혈청은 -20 ° C.에서 수집된 살균 - 여과 및 저장 </li></ol> 항원 제시 세포의 준비 <ol><li> 멸균 악기를 사용하여 참수 암흑의 thymus를 타고 얼음 PBS가 들어 페니실린과 스트렙토 마이신 (PBS - PS)에 놓으십시오. </li><li> 조직 문화 후드에 thymus을 가지고, 혈전 및 기타 오염 조직에서 청소하고 10 ML 포함된 10cm 배양 접시에 놓인 셀 스트레이너 (피셔 # 08-771-2) PBS를로 작은 조각으로 잘라 PS. </li><li> 각 조각은 살균 한 ML 주사기의 플런저의 뒷면을 사용하여 셀 스트레이너를 통해 누르면됩니다. 얼음에 50 ML 튜브에 단일 세포 현탁액를 수집합니다. </li><li> PBS - PS의 20-40보다 ML과 셀 스트레이너 린스. 1,250g에서 현탁액을 원심 얼음에 PBS - PS의 펠렛, 5 ML / thymus을 resuspend. </li><li> 감마 irradiator이 중지 (30 쥐)을 비추다 즉시 50 ML PBS - PS와 함께 두 번 씻는다. 참고 : 가장 좋은 thymus 기증자 나이 젊은 성인 쥐, 5-7주 있습니다. 최대 10 주 쥐를 사용하지만 세포의 숫자는 시간이 감소됩니다. 어떤 사람들은 남성보다 thymus 기증자는 것을 말하고 있지만 성별은 중요하지 않습니다. 모든 동물이 같은 방에 보관되어 있기 때문에 우리는 항상 여자를 사용하고 우리가 같은 방에있는 남성과 여성을 싶지 않아요. </li></ol> 자극 <ol><li> 일단 T lymphocytes 씻으십시오 그들을 계산합니다. </li><li> 항원 제시 세포를 계산합니다. </li><li> 믹스 3 X 10 6 ovalbumin 특정 자극을 매체 10 ML을 포함 10cm 배양 접시에있는 150 X 10 6 APCs와 T 세포. </li><li> 37 48 시간 동안 품어 ° C. </li></ol> 2. ovalbumin 특정 T의 lymphocytes의 입양 전송 <ol><li> 우리가 수신자로 사용 쥐들이 70-10주 오래된 암컷입니다. </li><li> T lymphocytes 세어, 그들은 크고 둥근해야합니다. 항원 제시 세포는 죽게 될 것입니다. </li><li> T lymphocytes을 원심 분리기 10 X 10 6 세포 / PBS에 ML에서 그들을 resuspend. </li><li> 3 ML 주사기 23 G 1 &quot;바늘을 사용하여 한 ML의 intraperitoneally을 주사. </li></ol> 3. 귀에 항원과 도전 <ol><li> 이틀 입양 전송 후, 다른 귀 한 귀로와 생리 (또는없는 항원)에 1 MG / ML ovalbumin 솔루션 (= 관련 항원) 20 μl를 주입. 당신이 염증 반응을 강화 합산을 잃을 수 있으므로 항원 솔루션을 필터링하지 마십시오. 참고 : 쥐나는 움직임을 피하기 위해 anesthetized해야합니다. 당신이 오른손잡이 경우, 왼쪽 엄지와 검지, 귀 안쪽되는 검지 손가락과 직면하고있는 쥐를 사이의 귀를 잡아. </li><li> 27G ½ &quot;바늘을 사용하여 귀의 날개에있는 솔루션을 주사. 같은 장소에서 정확하게 모든 쥐를 삽입하는보십시오. </li></ol> 4. DTH 측정 <ol><li> 귀는 빨간색과 염증의 징조로서 훌륭한 회전을 시작합니다. 용수철 마이크로 미터 (예 : 모델 Mitutoyo에서 PK - 0505)을 각 (内耳)의 두께를 측정합니다. </li><li> 각 시간 시점에서 각각의 귀에 대해 5-6 측정하고 평균을 계산합니다. </li><li> 최대 붓기 24 72 시간 사이에 발생한다. </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>

antigenic의 도전도 다시 (2) 피부에 예를 들어, 다른 사이트에서 수행할 수 있습니다. 우리는 그러나 그 도전 찾으십시오. 본 동영상에 표시된 귀의 날개를 사용하면 DTH 반응의 가장 정확한 측정을 위해 수 있습니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<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>
</tr>
<tr>
<td>Syngeneic rat serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1% final. See explanation in Protocol.</td>
</tr>
<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

지연 유형 과민성 (DTH)은 면역 시스템이 준비하는되어있는 대한 항원의 주사의 사이트를 침투 CCR7 - 효과기 기억 T의 lymphocytes로 인한 염증 반응이다. 염증 반응은 antigenic 도전의 사이트의 빨갛게과 팽창이 특징입니다. 그것은 면역 억제제의 생체내 효능에 결정하는 편리한 모델입니다. 피부 DTH는 항원에 특정한 T의 lymphocytes의 입양 전송 또는 항원, 그리고 주어진 피부 영역에서 염증 반응을 유발하는 항원과 이후의 intradermal 도전과 적극적인 예방 접종을 통해 유도된 수 있습니다. DTH 반응이 예제 ovalbumin, 투베르쿨린,​​ 파상풍 toxoid, 또​​는 열쇠의 림펫 hemocyanin에 대한 다양한 항원을 유도하실 수 있습니다. 이러한 반응도 MBP 여러 경화증 (1) 동물 모델 실험 면역 encephalomyelitis 유도와 쥐의 기본 단백질 (MBP)을 myelin 예를 들어, autoantigen에 대한 유발 수 있습니다. 여기 루이스 쥐에서 입양 DTH 반응을 유도하는 방법을 보여줍니다. 우리가 먼저 체외에서 ovalbumin 특정 T 세포를 자극하고 순진한 쥐 이러한 활성 셀 intraperitoneally를 삽입합니다. 다른 귀 윌은 생리 수신하는 동안 세포가 2 일간 생체내에서 평형 수있게되면, 우리는 귀 한쪽의 날개에 ovalbumin와 쥐에 도전합니다. 염증 반응이 3~72시간 나중에 표시되며, 귀 두께는 DTH 심각의 표시로 측정됩니다.

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

지연 유형 과민성 (DTH)는 CCR7 - 효과기 기억 T (TEM) lymphocytes로 인한 염증 반응이다. 여기서 우리는, 항원 특정 TEM 세포를 활성화 루이스 쥐에서 입양 DTH를 유발하고 염증 반응을 모니터링하는 방법을 보여줍니다.

고양이의 입양 지연 유형 과민성의 유도 및 모니터링

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 조회수.  University of California, Irvine (UCI). 지연 유형 과민성 (DTH)는 CCR7 - 효과기 기억 T (TEM) lymphocytes로 인한 염증 반응이다. 여기서 우리는, 항원 특정 TEM 세포를 활성화 루이스 쥐에서 입양 DTH를 유발하고 염증 반응을 모니터링하는 방법을 보여줍니다.

비디오: 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.

쥐에서 Active 지연 유형 과민성 (DTH)의 유도 및 모니터링

연구

생물학

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.

입양 DTH의 유도 후 귀에 영상 효과기 메모리 T 세포

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.

칙의 배아에서 신경 유도에 대한 분석

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.

쥐에서 유선의 형태론과 유방암 위험의 변화

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.

세포 배양의 유도와 Hypoxia의 시험

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.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

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.

갑각류와 장기 치명적인 독성 시험 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.

간엽 전환을 유도하고 상피의 분석

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.

체외 합성에 인간의 세포에서 단백질 발현 유도의 mRNA를 수정의

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.