저자는 스웨덴 연구 협의회, 전략 연구, 스웨덴의 암 사회, 스웨덴의 아동 암 사회 아케 Wibergs 재단과 Jeanssons 기초에 대한 스웨덴의 기초에 의해 지원됩니다.

1. 세포에 프로브를 소개합니다 <ol><li> 안달 기반 프로브 및 플라스미드 부여 저항 공동 transfect 세포. 관심의 셀 타입에 효율적입니다 transfection 방법을 선택합니다. U2OS 전지, 표준 칼슘 인산염 transfection 방법은 적절한 결과 15 줄. </li><li> 적어도 7 일 동안 적절한 항생제의 세포를 선택합니다. 프로브 및 제한 심각하게 세포주기에 영향을 미칠 독성 표현 수준이나 표현 수준과 세포의 크기를 표현하는 세포의이 풍요롭게의 금액입니다. </li><li> (선택 사항) 안정적인 클론을 선택하십시오. </li><li> 고정 또는 라이브 세포를 사용, CFP 및 YFP 모두 거의 같은 비율로 모든 세포에 존재하는지 확인합니다. </li><li> 일부 세포는 CFP 또는 YFP를 포함하는 경우에는 재조합 가능성이 높습 발생했습니다. 재조합은 박테리아 성장하는 동안 또는 프로브의 transfection 후 중 발생할 수 있으며, 후자의 경우에는 플라스미드 품질에 따라 달라질 수 있습니다. 플라스미드 P를 반복배상하고 문제가 지속되면 transfection하기 전에 플라스미드를 linearize. </li></ol> 2. 모니터링 ratiometric이 무서워 <ol><li> 챔버의 장착을위한 유리 바닥 요리, 또는 커버 전표에 씨앗 세포. 요리 / 커버 전표가 더 1.5 (170 μm의 두께)입니다 없는지 확인합니다. </li><li> 37 ° C. 설정 온도 제어와 동력 epifluorescence 현미경에 페놀 붉은없이 성장 매체 전지를 탑재 중 CO 2 또는 2 독립 미디어 등 Liebowitz - 15 CO를 사용하여 미디어의 산도 조절을 보장합니다. </li><li> 전송 조명을 사용하여 세포에 초점. 형광등을 사용하는 전지를 보지 마라. </li><li> YFP 여기 (YFPex)와 YFP 방출 (YFPem) 필터 및 적절한 이색성 거울을 사용하여 12 또는 16 비트 이미지를 획득하여 시작합니다. 여전히 필요한 subcellular 해상도를 허용 가능한 최대한의 binning을 사용합니다. 노출 시간이나 강도를 감소 높은 binning을 사용하면 phototoxicity을 피하기 위해 중요합니다. </li><li> 나를asure 또는 셀 찾아볼 수없는 영역에서 최소 거친 평균 및 최대 픽셀 값을 측정 또는 추정하여 배경 강도와 대략적인 소음을 예상하고있다. 변경 노출 시간 및 중성 밀도 필터 때문에 세포의 평균 신호는 약 배경 강도 플러스 5-10 회 맥스와 분 배경 농도 차이입니다. </li><li> 포화되지 가까이하지 않는 이미지를 확인합니다. 이미지의 최대 강도는 세포가 유사 분열을 입력하면 강도 차이 있도록 동적 범위 (12 비트 이미지에서 예를 들어 최대 2000)의 절반 미만이어야한다. </li><li> CFP의 여기와 YFP 방출 필터 및 적절한 이색성 거울을 사용하여 단계 2.4-2.6를 반복합니다. </li><li> 2.7 점 2.4에 사용되는 지역 제외, transfected 세포 여러 영역을 선택하고 최대 강도를 보장은 모든 세포의 다이나믹 레인지의 절반 이상이 적습니다. </li><li> 두 이미지 모두 4 획득 시간 경과 실험을 시작합니다YFPex를 사용하여 5 분 - YFPem 및 CFPex - YFPem 필터 설정합니다. </li></ol> 3. 조건을 확인하고 최적화 <ol><li> 획득한 이미지를 열고 유사 분열에서 transfected 프로브를 표현하는 세포 유사 분열하는 세포주기 시간을 모니터링합니다. </li><li> 사용되는 세포 유형에 게시된 데이터에 측정된 세포주기 시간을 비교합니다. 또한, 영화가 참조 셀 사이클 시간을 얻을에만 전송 빛의 하나의 이미지 (예 : DIC 또는 위상 대조) 매 시간을 인수함으로써 세포를 untransfected. </li><li> 유사 분열에서 유사 분열하는 시간이 세포주기의 타이밍을 참조에 해당하는 경우에는 4 단계로 진행합니다. 또는 더 노출을 사용하여 다시 취득 이미지, 더 많은 시간을 포인트 또는 여러 개의 Z - 레벨과 단계를 반복합니다 3. </li><li> 세포주기의 타이밍을 참조에 해당하지 않는 유사 분열에서 유사 분열하는 시간 경우에는, 영화는 결국에서만 전송 빛의 하나의 이미지 (예 : DIC 또는 위상 대조) 하나의 이미지 뒤에 매 시간을 인수함으로써 세포를 transfectedtransfected 세포를 식별하는 실험. </li><li> transfected 세포가 아직 이상 세포주기 시간을 보여주면, 1 단계를 반복하지만 적은 프로브를 transfect 또는 탐침의 매우 낮은 금액을 포함하는 안정적인 클론을 선택하십시오. </li><li> transfected 세포가 형광 불빛이없는 정상적인 세포주기 시간을 표시하는 경우, 이미지 사이의 binning, 중성 밀도 필터, 노출 시간 및 시간을 수정하여 2 단계지만, 낮은 노출을 반복합니다. </li></ol> 4. 초조 Analysing <ol><li> 분석은 현미경 전용 대부분의 소프트웨어로 수행할 수 있습니다. 여기서 우리는 프리웨어 ImageJ (사용 분석 수행하는 방법을 설명합니다 <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij을</a> 플러그인 비율 플러스 (활용) <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus를 . HTML</a> ). </li><li> ImageJ에서 이미지를 엽니다. 경우에는 귀하의 이미지는 여러 가지 빛깔의의 stac의 형식에K (예 Deltavision) 먼저 hyperstack로 변환하여 각 채널을 별도 : hyperstack에 이미지 hyperstack - 스택과 이후를 사용하여 단일 채널 스택에 분할 : 차원을 이미지 hyperstack - 줄이기 </li><li> 그것만이 비율이 0과 1 사이의 범위에하지 않는함으로써 이미지의 시각적 선명도를 증가하기위한 것일 뿐이므로이 단계는 선택 사항입니다 프로세스 수학 곱하기 : 3 이용한 YFPem 스택 - YFPex을 곱하면됩니다. </li><li> YFPex에 - YFPem 스택, 세포의 결여된하는 지역에 관심을 (ROI)의 영역을 그리지만, 그 측정이 될 수있는 세포에서도 가까운 거리에 있습니다. 배경 및 신호 강도가 모두 일반적으로 이미지의 중앙에 강력한는 같은 이미지의 다른 세포가 다른 지역을 요구할 수 있습니다. </li><li> 투자 수익 (ROI) 관리자에게 투자 수익 (ROI)를 추가 분석 - 도구 - 투자 수익 (ROI) 관리자. </li><li> 분석 - 측정 : - YFPem와 CFPex - YFPem 스택 YFPex 모두 투자 수익 (ROI)의 평균 강도를 측정 </em&gt;. </li><li> 여러 timepoints에서 반복하고 관심 세포에 가까운 배경 농도가 영화 전반에 걸쳐 유사 확인합니다. </li><li> 분석 - 설정 측정 - 최소 및 최대 회색 값 : 최소한의 강도를 포함하는 측정을 설정합니다. </li><li> 세포의 대부분을 다루는 관심 영역을 그려과 YFPex 모두 최소 농도 측정 - YFPem와 CFPex - YFPem 스택을. 4.6에서 측정한 최소 강도와 배경 강도의 차이를 가지고 두하여 나눕니다. 이것은 클리핑의 가치에 대한 시작을 예측할 수 있습니다. </li><li> 오픈 비율 플러스. YFPem 및 CFPex - - YFPem 스택 YFPex을 선택합니다. 비율을 선택 CFPex 걱정 위해 - 스택 1로 YFPem - 스택 1로 YFPem을 거꾸로가 인산화시 효율 저하를 걱정 프로브 선택 YFPex를 시각화 비율을 걱정하십시오. </li><li> 측정된 배경 농도 및 클리핑 값을 넣습니다. </li><li> 결과 비율 스택의 배율을 설정하고 적당한 모양의 U를 적용비율 변화를 시각화하는 P 테이블 (LUT). </li></ol> 5. 대표 결과 Plk1 활동은 유사 분열 동안 G2 단계와 봉우리의 핵에서 처음 볼 수 있습니다. 그림 3은 제 2에서 설명한대로 최소한 phototoxicity 설정을 사용하여 실험을 보여줍니다. 이것은 대표적인 초기 결과주의와 이미지 사이의 노출 조건이나 시간이 잡음 비율 또는 시간적 해상도로 신호를 증가 수정할 수 있습니다하시기 바랍니다. 프로브를 표현 세포의 대부분은 이미징 조건과 프로브의 표현 수준은 세포주기의 타이밍에 영향을 미치지 않습니다 나타내는 20 사이 및 25 시간의 세포주기의 시간 분아 따위에 의해 중식 그 그림 3 차원 보여줍니다. 상당한 소음이 있지만, Plk1 활동의 추세는 G2의 증가와 유사 분열 14 peaking하면 (그림 3A) 명확하게 볼 수 있습니다. 이곳으로 원시 데이터를 처리하는 말은 평균 거꾸로 RA의 kymograph (그림 3B) 또는 부량로 제시 필터링<html> 티오 (그림 3C)는 명료을 강화할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3410/3410fig1.jpg" /> 그림 1. 무서워 기반 프로브의 원리 키나제와 인산 가수 분해 효소 활동을 모니터링합니다. 두 fluorophores, 일반적으로 CFP (파란색)와 YFP (녹색)은, phospho - 결합 도메인 (오렌지색)과 phosphorylatable 시퀀스 (노란색)에 의해 연결되어 있습니다. 따라서 탐사에 conformational 변화를 유도 phospho 결합 도메인에 바인딩 인산화 (적색) mediates. CFP와 YFP 사이의 효율성을 걱정에 영향을 미치는 두 fluorophores, 사이의 거리 또는 방향의 차이에 conformational 변화 결과. 안달 흥분 CFP 및 모니터링 YFP 방출 (점선)에 의해 시각하실 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3410/3410fig2.jpg" /> 그림 2. 실험 절차의 도식 개요. p_upload/3410/3410fig3.jpg &quot;/&gt; 그림 3. Plk1 활동은 유사 분열 동안 G2 단계와 봉우리의 핵에서 처음 볼 수 있습니다. 안달 기반 프로브 모니터링 Plk1 활동 9,14을 표현 U2OS 세포는 20x NA 0.7 공기 객관적이고 수은 램프를 갖춘 Deltavision Spectris 이미징 시스템을 사용하여 60 시간에 대한 촬영했다. 이미징 조건 4X binning과 중성 밀도 필터를 사용하여, 같은 섹션 2에 명시된 최소한의 phototoxicity를 일으킬 선정되는 들어오는 빛을 차단 99%. 네 부문을 통해 한 셀 아래 거꾸로 걱정 - 비율, 잘못된 색상 표현. B, 의미 필터를 적용한 후에에 표시된 셀의 kymograph. C, 거꾸로의 부량는 A. D에 표시된 셀, 50의 누적 mitotic 항목의 비율을 걱정 딸 세포의 부서를 포함하여 표현 세포, - 프로브를 무서워.

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Biol. 189, 247-259 (2010).</li><li>Macurek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polo-like+kinase-1+is+activated+by+aurora+A+to+promote+checkpoint+recovery.">Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.</a> Nature. 455, 119-123 (2008).</li><li>van der Eb, A. J., Graham, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+transforming+activity+of+tumor+virus+DNA.">Assay of transforming activity of tumor virus DNA.</a> Methods. Enzymol. 65, 826-839 (1980).</li><li>Lamprecht, M. R., Sabatini, D. M., Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+free,+versatile+software+for+automated+biological+image+analysis.">CellProfiler: free, versatile software for automated biological image analysis.</a> Biotechniques. 42, 71-75 (2007).</li><li>Roszik, J., Lisboa, D., Szollosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intensity-based+ratiometric+FRET+in+image+cytometry--approaches+and+a+software+solution.">Evaluation of intensity-based ratiometric FRET in image cytometry--approaches and a software solution.</a> Cytometry A. 75, 761-767 (2009).</li></ol>

우리는 공개 아무것도 없어.

세포주기에 걸쳐 무서워 모니터링하는 것은 외부 자극에 단기 반응을 평가했을 때 덜 중요한 고려 사항이 필요합니다. 첫째, 세포주기의 진행은 쉽게 phototoxicity이 최소로 보관이 필요한, ​​스트레스 신호에 의해 불안정하게된다 는걸 알수 있습니다. 둘째, 모든 기자 잠재적 kinases, phosphatases 또는 상호 작용 도메인을 titrating으로 세포 프로세스에 영향을 줄 수 있습니다. 실험 조건이 충분한 경우...

<table border="1"><tbody><tr><td> 시약 </td><td> 카탈로그 번호 </td><td> 회사 </td></tr><tr><td> Leibovitz L - 15, 아니 페놀 레드 </td><td> 21083-027 </td><td> GIBCO, 생명 기술에 의해 </td></tr><tr><td> DMEM + Glutamax - I </td><td> 31,966 </td><td> GIBCO, 생명 기술에 의해 </td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0.05 % 트립신 EDTA (에틸렌 다이아 민 테트라 초산) </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14,287 </td><td> GIBCO, 생명 기술에 의해 </td></tr><tr><td> Puromycin </td><td> P8833 </td><td> 시그마 - 올드 리치 </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

포스터 공명 에너지 전달 (무서워) 기반 기자 1 살아있는 세포의 내생 키나제와 인산 가수 분해 효소 활동의 평가를 허용합니다. 이러한 프로브는 일반적으로 phosphorylatable 시퀀스와 phospho - 바인딩 도메인 방해 CFP 및 YFP의 변종의 구성됩니다. 인산화되면 변화로 이어지는 CFP와 YFP 사이의 거리 또는 방향의 변경 결과 프로브 변경 형태는 (그림 1) 효율성을 무서워. 몇몇 프로브는 PKA 2, PKB 3, PKC 4, PKD 5, ERK 6, JNK 7, CDK 18, 오로라 B 9 기자와 Plk1 구 등의 여러 kinases와 phosphatases의 활동 균형을, 모니터링, 지난 10 동안 공개되었습니다 . 모듈형 설계 감안할 때, 추가 프로브는 향후 10 등장할 가능성이있다. 세포주기를 통해 진행은 스트레스 signali에 의해 영향을받습니다NG 11 경로. 특히, 세포주기는 세포가 스트레스를 12 회복하는 경우에 비해 성장을 교란되지 않은 동안 다르게 규제입니다. 세포주기를 통해 세포의 시간 경과 이미징 따라서 특별한주의가 필요합니다. ratiometric 이미징을 채용 특히 잡음 비율 높은 신호를 두 개의 이미지가 정확하게 결과를 해석하는 데 필요한 때문에 이것은 문제가된다. Ratiometric는 키나제와 인산 가수 분해 효소 활동에 세포주기에 의존 변경 이미징이 predominately 세포주기 8,9,13,14의 하위 섹션에 제한되었습니다 무서워. 여기, 우리는 인간의 세포주기에 걸쳐 ratiometric 이미징을 사용하여 걱정 기반 프로브를 모니터링하는 방법을 논의합니다. 방법은 생명 과학의 많은 연구자에게 제공하고 현미경이나 영상 처리의 전문 지식을 필요로하지 않는 장비에 의존합니다.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

안달 기반 기자 점점 라이브 세포 키나제와 인산 가수 분해 효소 활동을 모니터링하는 데 사용됩니다. 여기 대상 인산화에서 세포주기 종속 변화를 평가하기 위해 안달 기반의 기자를 사용하는 방법에 대한 방법을 설명합니다.

안달 Ratiometric하여 세포주기를 통해 키나제와 인산 가수 분해 효소 활동을 모니터링

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

14.2K 조회수.  Karolinska Institutet. 안달 기반 기자 점점 라이브 세포 키나제와 인산 가수 분해 효소 활동을 모니터링하는 데 사용됩니다. 여기 대상 인산화에서 세포주기 종속 변화를 평가하기 위해 안달 기반의 기자를 사용하는 방법에 대한 방법을 설명합니다.

비디오: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Saphenous 베인 통해 심장 찔린로 쥐에서 혈액을 그리기

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

연구

생물학

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

포유 동물 세포의 세포주기의 위치 분석

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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 ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

Unimolecular 바이오 센서를 사용하여 라이브 세포에서 이벤트를 신호의 실시간 모니터링을위한 현미경을 안달

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

단일 분자 FRET에 의해 DNA 루핑을 공부

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

의 동기화<em&gt; Caulobacter Crescentus</em&gt; 세균 세포주기의 조사에 대한

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

실험 접근법은 미토콘드리아 현지화 및 핵 세포주기 키나제,는 Cdk1의 기능을 연구하는

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

G 단백질 선택적 G​​PCR의 입체는 라이브 셀 서스펜션 형광 분석에서 FRET 센서를 사용하여 측정

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

유사 분열 동안 다기능 세포 주기 단백질의 역할을 연구 하기 Mitotic 세포 동기화와 고해상도 Confocal 현미경 검사 법

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...