이 작품은 호주 국립 보건 의학 연구위원회 (NHMRC # 559007) 및 아동 당뇨병 연구 재단 (JDRF 4-2006-1025)와 빅토리아 정부의 운영 인프라 계획의 보조금에 의해 지원됩니다. 우리는 인간의 조직을 제공하는 톰 Mandel 작은 섬 이식 프로그램 작은 섬 절연 팀의 구성원을 감사드립니다. 인간의 조직이 지역의 윤리적 승인을 수집하여 사용되었다 (세인트 빈센트 병원 HREC-A 011 / 04, 세인트 빈센트의 건강 HREC-A 135 / 08).

1. 비장 조직을 준비 <ol><li> 참고 - 모든 인간 자료는 잠재적으로 전염성이 모든 절차는 클래스 II의 층류 내각에서 실시되어야한다로 취급해야합니다. 멸균 가위와 집게를 사용하여, 비장 섹션 (크기 ~ 1~2센티미터)에서 지방과 섬유 조직을 제거하고 가능한 한 바깥 쪽 캡슐 소재의 많은을 잘라. </li><li> 비장 조직의 작은 조각 (1~2센티미터 3) 잘라 멸균 50 ML 팔콘 튜브에 각각의 조각을 배치합니다. </li><li> 액체 N 2 침수에 의해 조직의 조각을 스냅 - 고정. </li><li> -80에서 저장 ° C. 유사한 프로토콜은 다른 조직 (들)에 적합합니다. </li></ol> 2. 스토리지에 대한 인간의 작은 섬 준비 <ol><li> CMRL 미디어 문화 섬. 10 ML 원뿔 바닥 관에서 섬을 수집하고 5 분을 위해 1,500 rpm으로 centrifuging하여 PBS로 두 번 씻어. PBS를 넣어하고 종이 타월에 잠시 역 튜브를 삽입하여 잔류 버퍼를 배수한다. 주의하지 말아라섬를 제거합니다. </li><li> -80에서 액체 질소와 매장에서 일단 배수, 재 캡 튜브와 스냅 - 동결 ° C. </li></ol> 3. 추출 준비 <ol><li> 4에 BAW 믹스 (10:30:60 % V / V) 및 매장 ° C. 준비 </li><li> -80 ° C.에서 튜브를 제거 실온에서 해동. </li><li> 조직의 조각을 충당하기 위해 충분한 얼음처럼 차가운 BAW을 추가합니다. 섬 3-5 ML을 사용하십시오. 비장 조직의 경우 조직의 조각의 크기에 따라 10-20 ML을 사용합니다. </li><li> 조직 균질를 조립. 70 % 에탄올 / 물 10-20 ML &#39;을 homogenizing&#39;로 청소하십시오. </li><li> 조직 및 BAW 솔루션 관에 균질 프로브를 삽입 한 후 여러 뿜어의 조직을 균질화. 이후 얼음 양동이에 관세요. </li><li> 철저히 10-20 70 % 에탄올 / 물 ML 후 BAW 버퍼를 homogenizing하여 샘플 사이의 균질를 청소. 이것은 우리 한 후 70 % 에탄올 / 물 samples.Dismantleand 깨끗한 사이의 조직의 교차 오염을 방지5. </li><li> 만 수용성 물질을 포함하는 추출물이 필요한 경우 10 분 동안 RT에서 4,000 rpm으로 균질 조직 추출물을 원심 분리기. 더 많은 원유 추출물가 필요한 경우, 5 분 동안 RT에서 1,000 rpm으로 회전. BAW-불용성 단백질을 추출하기위한 가장 적합한 기술은 단백질의 하류 분석에 따라 달라집니다. 기능 면역의 assays에 사용하기 위해 우리는 우리가 이전에이 잘 6 용납 할 수 발견으로 8 M의 요소에 불용성 일부를 분해하려고 시도하는 것이 좋습니다 것입니다. </li><li> 깨끗한 튜브에 표면에 뜨는 전송과 얼음에 넣어. </li><li> BCA 분석 또는 이와 유사한을 사용하여 추출물의 단백질의 농도를 결정합니다. </li></ol> 4. 건조 추출물을 동결 <ol><li> 단백질 필수의 질량 (예, 튜브 당 100 μg)에 따라 적절하게 homogenate를 희석하여 표시 5.0 ML 멸균, 매 (12x75 mm) 튜브에 aliquots을 투여. 우리는 자주 100 μg / 튜브를 사용합니다. </li><li> 1을 사용각 튜브의 뚜껑에 3 개의 구멍을 만들 8-20 게이지 멸균 주사기 바늘. </li><li> ~ 10 분에 드라이 아이스 나&gt; 1 시간에 -80 ° C 냉장고에 그들을 배치하여 두 튜브를 고정. -80에서 저장 ° C lyophilizer에 넣어 준비가 될 때까지. </li><li> 냉동 건조기에 전환 (-100 ° C) 평형 할 수 있습니다. 이 약 30 분 정도 소요됩니다. </li><li> 볼륨에 따라 냉동 건조는 3 시간 이내에 완료하지만 우리는 정기적으로 하루 아침에 우리의 샘플을 떠날 수 있습니다. </li><li> 건조 사이클을 완료 한 후, 진공 펌프를 끄고 천천히 챔버로 압력을 수 있습니다. 랙을 제거합니다. </li><li> 멸균 후드에서 튜브에서 구멍 마개를 제거하고 새 (매 352,032)로 교체하십시오. </li><li> -20 ° C.에 상점 튜브 샘플은 문화 미디어, 또는 다른 버퍼에 재구성하고, 함수 또는 생화학 assays에서 사용할 수 있습니다. </li></ol> 5. 대표 결과 그림 1은 단백질의 착색을 보여줍니다젤 비장 및 췌장 고갈 췌장 조직 (acinar 표시)에서 추출물과 인간의 섬을 (라벨 섬) 정화와 함께로드. 결과는 각 조직에 대해 서로 다른 분자량의 단백질의 좋은 표현을 보여줍니다. 인간 T-세포 증식을 자극하는 조직 추출물의 용량은 CFSE 기반 확산 분석 7 (그림 2)를 사용하여 테스트되었습니다. 이 분석에 사용 된 PBMC는 제 1 형 당뇨병과 개인으로부터 격리되었다. 응답의 크기는 항원이없는 5000 CD4 +, CFSE 밝은 세포 당 CFSE 흐린 세포의 수의 비율로 표시됩니다 : 5,000 CD4 당 CFSE 흐린 세포의 +, 세중의 샘플 7 항원과 CFSE 밝은 세포. 결과는 약한를 표시하지만, acinar에 대응 (CD1 = 3.5)와 작은 섬 추출물 (6.8)에 강한 반응 감지, 증식. Inactivated 인플루엔자 바이러스 (CDI = 142.6)이로 포함되어 있습니다긍정적 인 제어 할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> 그림 1. 단백질 젤. <img alt="그림 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> acinar과 작은 섬 추출물에 대한 CFSE 기반의 확산 분석에서 그림 2. 결과. <a href="http://www.jove.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> .

<ol>
	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

관심 없음 충돌이 선언 없습니다.

우리는 이러한 세제 등 독성 화학 물질에서 자유를 인간의 조직에서 추출을 생성 싶었 기 때문이 프로토콜은 개발되었습니다. 특히 우리는 체외에서 인간의 면역 기능의 assays에서 사용할 수있는 인체 조직의 추출물을 준비하는 데 사용했습니다. 이 프로토콜을 사용하여 준비 추출물은 똑같이 버퍼에 재구성 및 blotting 서쪽이나 액체 크로마토 그래피 등 여러 생화학 분석, 사용할 수 있습니...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 5 ML 12 X 75mm 멸균 폴리스티렌 튜브 </td><td> BD 팔콘 </td><td> 352054 </td><td></td></tr><tr><td> 튜브의 폴리스티렌 튜브에 캡 (위) </td><td> BD 팔콘 </td><td> 352032 </td><td></td></tr><tr><td> 50ml 멸균 튜브 </td><td> Becton 디킨슨 </td><td> 352070 </td><td></td></tr><tr><td> 아세토 니트릴 </td><td> Mallinckradt 화학 </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-좋았어요 </td><td> 시그마 알드리치 </td><td> 537993-IL </td><td></td></tr><tr><td> 균질 : PRO200 </td><td> 바이오 전략 </td><td> 01-01200 </td><td> 10 × 115mm 톱니 생성기 </td></tr><tr><td> Lyophilizer </td><td></td><td></td><td> Virtis, Benchtop 4K </td></tr><tr><td> 멸균 니들 18-20 게이지 </td><td> Becton 디킨슨 </td><td> REF 302,032 </td><td></td></tr><tr><td> CMRL-1066 매체 </td><td> 시그마 </td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td> 시그마 </td><td> D8537 </td><td></td></tr></tbody></table> 표 1. 특정 시약 및 장비.

extraction of tissue antigens for functional assays

B와 T 세포에 의해 인식 적응 면역 반응의 항원 대상, 대부분은 1 정의되지 않았습니다. 이 면역 질환과 암 2 특히 사실입니다. 우리의 목적은 면역 질병 1 형 당뇨병 1,3,4,5 인간의 T 세포에 의해 인식 항원을 조사하는 것입니다. T 세포에 의해 인식 항원이 발견되지 않는 조직에 대한 인간 T-세포 반응을 분석하기 위해 우리는 기능 assays 6 호환되는 형식으로 인간의 조직에서 단백질 항원을 추출하는 방법을 개발했습니다. 추출 방법은 인간의 말초 혈액 mononuclear 세포에 독성했다 세제를 포함하는 lysate를 얻을 수 있기 때문에 unpurified 조직 추출물로 이전, T-세포 반응을 측정 할 수 없습니다. 여기 인간의 T 세포에 독성이없는 형식으로 인간의 조직에서 단백질을 추출하기위한 프로토콜을 설명합니다. 조직은 butan-1-안녕, 아세토 니트릴 및 와트의 혼합물에 균질하고 있습니다어 (BAW). 조직 추출물의 단백질 농도는 측정 및 단백질의 알려진 질량은 튜브에 aliquoted 있습니다. 추출 후, 유기 용제는 lyophilization에 의해 제거됩니다. 필요 할 때까지 동결 건조 된 조직 추출물은 저장 될 수 있습니다. 면역 기능, 면역 세포의 정지 assays에 사용하기 위해, 적절한 문화 미디어, 동결 건조 추출물에 직접 추가 할 수 있습니다. 이 방법을 사용하여 준비 추출물에 대한 응답으로 PBMC의 시토 킨 생산 및 확산은, 쉽게 측정 하였다. 따라서, 우리의 방법은 T-세포 반응의 분석에서 항원의 소스로 사용할 수있는 인간의 조직 lysates의 급속한 준비를 할 수 있습니다. 이 방법은 이식, 암과 autoimmunity의 조직에 적응 면역 반응의 분석을 용이하게 할 것을 권장합니다.

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

기능 T-세포 assays에서 항원의 소스로 사용되는 인체 조직의 추출물을 준비하기위한 간단한 프로토콜은 설명되어 있습니다. 이 방법은 조직 유래 항원에 대한 T 세포 반응을 측정 할 수 있습니다<em&gt; 체외에서</em&gt;.

기능 Assays을위한 조직 항원의 추출

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

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10.8K 조회수.  St. Vincent's Institute of Medical Research. 기능 T-세포 assays에서 항원의 소스로 사용되는 인체 조직의 추출물을 준비하기위한 간단한 프로토콜은 설명되어 있습니다. 이 방법은 조직 유래 항원에 대한 T 세포 반응을 측정 할 수 있습니다 체외에서.

비디오: Extraction of Tissue Antigens for Functional Assays

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

마우스에 대한 진한 / 라이트 전환 테스트

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

연구

생물학

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidic Mesophases를 사용하여 구조 결정을위한 막 단백질 Crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

약리 및 기능성 유전자 Assays은 Planarian의 재생을 조작하는<em자포니카> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

유전자 표현 기계의 기능 Assays을위한 소규모 핵 추출물

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

문화의 세포에 대한 생존 분석 실험

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

분리 및 배양 세포 및 마우스 조직에서 미토콘드리아의 기능적 분석

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

RNA 및 DNA 추출 모두 포르말린 고정 파라핀 - 임베디드 조직 코어의 제조

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...