만성 - 원래대로 돌아가는 EAE의 유도 및 모니터링 유제는 보충 완료 인드의 도움이되는 추가 수용액에서 항원의 1:1 혼합물이다. 그것은 도움이되는가 아닌 다른 방법으로 라운드에 항원을 추가하는 것이 매우 중요합니다! 결정과 유제를 주입하면 폐기물의 많은있다. 항상 1.5 또는 2 배에게 필요한 것 이상을 준비. autoclaved 박격포, 유봉, 유리 주사기, 그리고 다리를 사용하십시오. 모든 플라스틱은 멸균되어야합니다. 준비는 정기적으로 실험실 벤치에서 할 수 있습니다. 1. 보충 도움이되는의 준비 Mycobacterium 결핵 (Difco 카탈로그 # 231141)와 절구에 장소 30 MG 나가는거야. 세균을 깨는 피하기 위해 너무 열심히 누르지 않고 얇은 가루로합니다. 프로 인드의 도움이되는 H37Ra (Difco 카탈로그 # 231131)와 혼합을 완료 10 ML을 추가합니다. 이 보충 도움이되는 지금은 4 MG / ML Mycobacterium의 결핵이 포함되어 있습니다. 튜브로 전송합니다. 2. 척수의 homogenate의 준비 -80에서 냉동 DA 쥐 (연령 / 성별 무관) 및 저장 ° C.에서 척추 코드를 수집 보충 도움이되는과 : 1시 1분를 (볼륨 무게) 혼합 충분해야 냉동 척추 코드를 달다. 면도날로 가능한 벌금으로 척추 코드를 말하다, 제 1에서 사용되는 박격포로 전송하고, 유봉와 붙여넣기를합니다. 3. 유제의 준비 <ol><li> 장소는 튜브에 프로 인드의 도움이되는을 완료 보충. 고속으로 소용돌이. vortexing 동안 드롭하여 척수 homogenate 드롭을 추가합니다. 5 분 더 척수의 homogenate의 모든 추가, 소용돌이. 혼합물은 밝은 분홍색으로 설정해야합니다. </li><li> 5 ML 유리 주사기에 감광 유제를 넣고 (피셔 카탈로그 # 14-825 - 17L) 18G 다리를 사용하여 다른 5 ML 유리 주사기에 연결합니다. 그것이 (5-10 분) 하드된다 때까지 다른 주사기에서 유제를 보내드립니다. 유제 5 개 이상 ML이 준비되면, 몇 가지 5 ML의 주사기는 큰 주사기를 사용하지 마십시오 사용합니다. </li><li> 유제는 3 주 동안 4 ° C에서 주사기에 저장할 수 있습니다. 나는 그것이 분해되지 있는지 확인하기 위해 사전에 적어도 12 시간 준비하는 것이 좋습니다. 그것은 두께 2 단계에서 별도의 안 유지됩니다. 색상은 노란색이나 베이지색이 빛을 하룻밤 변경됩니다. </li></ol> 4. 쥐를 예방 접종 <ol><li> 몇 분 동안 주사기에 감광 유제를 혼용하여 주사에 사용되는 주사기로 전송할 수 있습니다. 단기 마취 23G 바늘 3 ML Luer - 잠금 주사기를 사용하여 꼬리의 아주 기본에 subcutaneously 200 μl를 주입. </li><li> 수신자가 7~9주 오래된 여성 DA의 쥐입니다. 우리는 할런 - 스프 라그 돌리에서 우리 쥐를 얻을. </li><li> 쥐 두 번 매일 매일 관찰하고 무게가 있어야합니다. 임상 증상은 7-15일 유제의 주입 후 예상된다. </li></ol> 임상 채점 : 0 : 없음 질환 0.5 : 말초 미약 꼬리 1 : 미약 꼬리 2 : 가벼운 paraparesis, 운동 실조증 3 : 중간 paraparesis, 수시로 쥐 여행 3.5 : 한 뒷다리가 마비되어 다른 이동 4 : 전체 뒷다리 사지 마비 5 : 완전한 뒷다리 사지 마비 및 실금 6 : 죽어가는, 어려움 호흡은 먹지도 않고 마시지도 않습니다. 즉시 안락사.

CB와 KGC는 Airmid 주식 회사에 대한 공동 설립자 및 컨설턴트 아르

이 프로토콜에 대한 몇 가지 중요한 고려 사항이 있습니다. DA의 쥐의 유전자들은로부터 구입하는 종족에 따라 약간 다를 수 있습니다. 이러한 차이는 유도를 EAE하는 쥐의 자화율를 증가 또는 감소됩니다. 당신의 쥐가 EAE에 더 민감한 경우가 아닌 보완 완료 인드의 도움이되는 또는 불완전한 프로 인드의 도움이되는을 사용하여 예방 접종의 강도를 줄이는 것이 좋습니다. 당신은 또한 유제를 준비하기 전에...

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Complete Freund's Adjuvant</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF 311-360-5</td>
<td>Manufactured by Difco</td>
</tr>
<tr>
<td>Mycobacterium tuberculosis H37Ra</td>
<td>Reagent</td>
<td>Fisher</td>
<td>DF3114-33-8</td>
<td>Manufactured by Difco</td>
</tr>
<tr>
<td>DA rat</td>
<td>Animal</td>
<td>Harlan Sprague Dawley</td>
<td>&nbsp;</td>
<td>Females, 7-9 weeks old.</td>
</tr>
<tr>
<td>Glass syringes - 5 ml</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-823-10 B</td>
<td>&nbsp;</td>
<tr>
<td>Metal bridge - 18G</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-825-17L</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lok plastic syringes - 3 ml</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>Needles - 20G 1 1/2</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-826-5C</td>
<td>&nbsp;</td>		</tbody>
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induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

다중 경화증 (MS)는 일반적으로 젊은 성인에 영향을 미치는 중추 신경계 (CNS)의 만성 염증성 질환입니다. 이것은 두뇌 및 척수에 전파 영역에서 demyelination과 glial 무섭게이 특징입니다. 이러한 병변은 신경 전도를 변경하고 CNS (예 : paraparesis, 마비, 실명, 실금)에 demyelinated plaques의 위치와 다릅니다 해제 신경 적자를 유발. 실험 면역 encephalomyelitis (EAE)은 MS의 모델입니다. EAE 먼저 토끼 척수 코드에서 성장 바이러스를 사용하여, 광견병 예방 접종 기간 동안 인간의 실수로 유도했다. inactivated 바이러스에 주입 척수의 잔류물은 CNS 질환을 유도. 이러한 관찰 다음, EAE의 첫 번째 모델은 하천에 의해 homogenate 및 1935 년에 Schwenther CNS와 함께 예방 접종을하지 않은 인간 영장류에서 설명했습니다. EAE 이후 인류의 다양한 생성되었고 종 / 변형에 따라 그리고 항원이 사용 immunizing 다른 과정을 따를 수 있습니다. 동일한 항원은 실험용 돼지의 만성 질병을 유도하면서 예를 들어, 도움이되는와 유제에서 기본적인 myelin 단백질과 루이스 쥐나 immunizing 것은 EAE의 급성 모델을 유도. EAE 모델 여기에 설명된 내용은 전체 프로 인드의 도움이되는에있는 유제의 DA 랫 척수에 대한 검찰의 쥐가 immunizing에 의해 유도됩니다. 쥐들이 7~14일 사후 예방 접종 이내 오름차순 이완된 마비를 개발할 수 있습니다. 임상 증상은 몇 주 동안 원래대로 돌아가는 - remitting 과정을 따르십시오. 병리학은 CNS와 demyelination의 plaques 큰 면역 infiltrates를 보여줍니다. EAE과 동물 돌보기위한 특별 고려 사항은 비디오의 끝부분에 설명되어 있습니다.

Watch this Scientific Journal Video about Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis at JoVE.com

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

DA 쥐에서 만성 - 원래대로 돌아가는 실험 면역 encephalomyelitis :이 비디오는 여러 경화증의 동물 모델의 유도 및 임상 채점을 보여줍니다. 전체 쥐의 척수하고 완전한 프로 인드의 도움이되는가 포함된 유제와 immunizing 쥐가 유도된 질병은, 인간의 질병을 닮은 임상 징후를 보여줍니다.

유도 및 만성 - 원래대로 돌아가는 실험 면역 Encephalomyelitis 임상 채점

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

induction-clinical-scoring-chronic-relapsing-experimental-autoimmune

Research

JoVE Journal

Biology

17.9K 조회수.  University of California, Irvine (UCI). DA 쥐에서 만성 - 원래대로 돌아가는 실험 면역 encephalomyelitis :이 비디오는 여러 경화증의 동물 모델의 유도 및 임상 채점을 보여줍니다. 전체 쥐의 척수하고 완전한 프로 인드의 도움이되는가 포함된 유제와 immunizing 쥐가 유도된 질병은, 인간의 질병을 닮은 임상 징후를 보여줍니다.

비디오: Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Experimental Approaches to Tissue Engineering

조직 공학 실험 접근법

연구

생물학

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.

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 ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

작업 워킹 레더 렁 : 채점 시스템과 실용적인 프로그램입니다.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNA의 중재 진 입을와 결합 라이브 세포 칼슘 영상은 CA를 나타냅니다 2 + ER 막의 누출 채널과 규제 메커니즘

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

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 ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

고양이와 쥐의 Ovariectomy 및 17β-estradiol 대체 : 비주얼 데모

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

3 차원 형광 이미지에서 세포 형태학의 변경 사항 단정 지을 분석 도구

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

손쥐 심방과 심실 Cardiomyocytes의 절연 및 KV 채널 레코딩

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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 ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

전기 영동 이동성 분석 (EMSA) 및 Shift 키를 사용하여 기능 비 코딩 유전자 변형에 대한 심사 DNA 화성 강수 분석 (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...