우리는 CPRIT (RP100119) 및 웰치 재단 (I-1665)의 자금 지원을 인정합니다.

전체 프로토콜은 약 사일 소요됩니다. 1. siRNA를 및 기자 형질에 대한 세포의 준비 우리는 여기에서 설명을위한 게놈 전체의 siRNA 라이브러리에서 siRNAs에의 작은 집합 (하나의 유전자를 대상으로 배 siRNAs에 구성된 각 풀에 96도 형식으로 384 가지의 siRNA 풀 배열)을 사용하여 siRNA의 라이브러리를 심문하기위한 프로토콜을 소개합니다. 이 특정 연구를 위해, 우리는 HCT116 비정상적인 Wnt의와 크라스 신호를 전시 세포와 p53의 활동을 이용합니다. 관심의 다른 세포 프로세스를 심문을 목표로 연구에 적절한 셀 라인은 식별 유사한 방식으로 평가해야합니다. <ol><li> 2 % 철을 함유 Dulbecco의 수정 이글 배지 (DMEM) 10 mL로 중화 한 다음 트립신 솔루션 / 판 1 ㎖를 사용하여 트립신에 의해 PBS 다음 수확 세포와 80 % ~ 90 % 합류 HCT116 세포 5 × 10cm 2 판을 씻어탈 소 혈청 (FBS), 1 % 페니실린 / 스트렙토 마이신 / 판. </li><li> DMEM / 2 % FBS / 1 % 페니실린 / 스트렙토 마이신 10 ㎖에 50 ML 원뿔 튜브에 정지 세포를 전송하고 5 분 ~ 130 XG에서 centrifugate, 상층 액을 제거하고 다시 중단 세포 펠렛. </li><li> 셀 카운터와 세포 수를 계산 한 다음, 10 5 세포 / ml의 세포 솔루션을 만들어 다음 단계를위한 150 ML의 세포 현탁액을 유지합니다. 멀티 드롭 자동화 된 액체 디스펜서 (이제부터는 멀티 드롭)를 사용하여 96도 세포 배양 플레이트의 각 우물 판 (10) 4 셀 (100 μL). 세포 현탁액은 도금 12 × 96 웰 플레이트이 경우 충분합니다. </li><li> 5 % CO 2와 37 ° 인큐베이터에서 C에서 세포와 번호판을 품어의 transfection 혼합물을 준비하는 동안. </li></ol> 2. 기자 구문 재고 솔루션을 준비 <ol><li> 분리 고품질 기자는 표준 midiprep 키트 (NucleoBond 엑스트라 미디가 제작하여 사용하여 DNA를 구성그리고 Clontech 사)는 각 기자 1 MG / ML의 최종 농도로 DNA를 희석. </li><li> 기자는 p53의-FL 30 μL, 8X TCF-RL, Elk1-GAL4의 30 μL, 10 μL UAS-CL과 함께 DNA의 혼합을 달성하기 위해 H 2 O 4 μL의 30 μL를 결합하여 주식 솔루션을 구축의 100 μl를 준비 기자의 최종 1:1:1:0.2 비율입니다. 이 DNA 혼합 원액의 최종 DNA 농도는 0.3 ㎎ / ML입니다. </li></ol> 3. siRNA를 / DNA 형질 믹스를 준비 프로토콜의이 부분에서, 우리는 12 × 96 잘 접시를 형질에 대한 충분합니다 siRNA의 / DNA 형질 믹스를 준비합니다. siRNAs는 4 X 96 웰 플레이트의이 테스트 라이브러리, 우리는 세중의 각 siRNA의 풀을 형질 것입니다. 우리가 사용하는 형질 시약 Effectene (Qiagen의)이라고합니다. 우리의 손이 배양 세포에의 siRNA 및 DNA의 동시 배달을 위해 작동하는 전용 시약이다. <ol><li> 기자 구조 주식 졸의 80 μl를 희석3 μg / ML의 최종 농도로 DNA 솔루션을 달성하기 위해 EC 버퍼 (Effectene 키트)의 8 ML의 ution. 일반적으로, 즉시 사용을위한 충분한 DNA 솔루션 믹스를 준비합니다. </li><li> 96도 / 플레이트의 각 웰이 DNA 솔루션의 플레이트 (20) μL. 96 잘 플레이트의 수는 siRNA의 풀 테스트 할 얼마나 많은에 따라 달라집니다. 예를 들어,이 경우에 우리는 384 다른 siRNA의 풀을 평가하는 4 × 96 웰 플레이트가 필요합니다. </li><li> 테스트 할 siRNAs는 5 μM의 주식 농도에 있어야합니다. 하지 않으면, 그들은 복제가 도금 및 각 라이브러리와 연관된 권장 희석 버퍼를 사용하여이 농도로 희석 할 수 있습니다. </li><li> 잘 Biomek 자동 액체 처리기와 희석 DNA 주식을 포함하는 PCR 플레이트의 해당 각 5 μm의 siRNA의 주식의 2 μl를 전송합니다. 연구의 규모에 따라 멀티 채널 피펫은 충분합니다. </li><li> 이제, 우리는 DNA / siRNA의 PLA의 각 웰에 증강 용액 (Effectene 키트) 1 μl를 추가합니다테. 이것은 다시 자동 액체 핸들러 또는 멀티 채널 피펫으로도 수행 할 수 있습니다. </li><li> 각 well에 Effectene 형질 시약의 3 μl를 추가하고 추가로 10 분 동안 RT에서 품어 증강 반응이 5 분 RT에서 발생 할 수 있습니다. </li><li> 형질 복잡한 형성을 중지하려면, PCR 플레이트의 각 웰에 10 ㎕의 DMEM / 2 % FBS / 1 % 페니실린 / 스트렙토 마이신을 추가합니다. </li><li> 세포 (세포 배양기에서 검색)을 포함하는 96 잘 플레이트의 각 웰에 형질 믹스의 10 μl를 전송합니다. 각 형질은 세중의에서 수행되므로, 동일한 믹스 세포를 포함하는 두 개의 추가 96 잘 접시에 적용됩니다. </li><li> 36 시간 동안 5 % CO 2 가습 환경에서 37 ° C에 추가 형질 혼합물로 세포를 품어. </li></ol> 4. 세포 샘플에서 루시 페라 제 활동을 결정 36 시간 후, 루시퍼 활동 측정을위한 준비형질 세포합니다. 엔드 포인트 당 실험 방식에 의해 결정해야한다. 본 연구에서는 36 시간의 배양 시간 이전 결과 결정 의미를 충분히 강력한 신호를 굴복했다. 이 연구는 여러 기자 (세포 세포질에서 발현 배지, 두 사람으로 분비 한) 통합으로, 우리는 모두 배양 배지뿐만 아니라 세포 파쇄물에서 측정 값을 얻을 것이다. <ol><li> 문화 매체 CL 활동의 탐지 : <ol><li> 배양액 20 μL은 복제 도금 흰색 불투명 96 웰 플레이트 (중 Biomek 액체 처리기 또는 멀티 채널 피펫을 사용)합니다. </li><li> 각 well에 CL 분석 버퍼 20 μl를 (시스템 대상)를 추가합니다. </li><li> 각 well에 Cypridina 루시페린 기판 (시스템 대상)의 10 μl를 추가하고 luminometer (예를 들어 BMG에 의해 생성 된 Pherastar 플레이트 리더)를 사용하여 CL 활동을 감지합니다. </li></ol></li><li> FL 및 RL 활동 감지 : <ol><li> C를 용해먼저 문화 매체를 제거하여 ELL 학생. 이것은 거꾸로 판을 켜고 싱크 매체를 던지기에 의해 신속하게 수행 할 수 있습니다. 나머지 매체는 부드럽게 종이 타월에 거꾸로 판을 눌러 제거 할 수 있습니다. 플랫폼 로커에 멀티 드롭과 장소를 사용하여 세포의 각 웰에 1X 수동 용해 버퍼 (Promega 사)의 30 μl를 추가 RT에서 5 분 중간 속도를 설정합니다 (Bellco이가 만드는 한 회사입니다.) </li><li> luminometer를 사용하여 멀티 드롭 즉시 측정 FL 활동을 사용 루시 페라 제 분석 시약 II (프로 메가 듀얼 루시 페라 키트의 일부)의 20 μl를 추가합니다. 다음으로, 추가 및 중지 FL 활동을 담금질하고 RL 활동의 측정을 허용합니다 저런 형광 시약 (Promega 사). 참고 : 확장 된 신호 지속 시간 (예 : 프로 메가에서 이중 글로 루시 페라 키트 등)을 허용 기판 믹스를 사용하지 않는 플래시 키트의 사용은 기판뿐만 아니라시 신속한 측정 (5 분 이내)이 필요합니다. </li></ol></li></ol>

<ol>
	<li>Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., Kricka, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+and+bioluminescent+reporter+gene+assays.">Chemiluminescent and bioluminescent reporter gene assays.</a> Analytical Biochemistry. 219, 169-181 (1994).</li><li>Miraglia, L. J., King, F. J., Damoiseaux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+luminescent+reporter+gene+assays.">Seeing the light: luminescent reporter gene assays.</a> Combinatorial Chemistry &amp; High Throughput Screening. 14, 648-657 (2011).</li><li>McGuire, S. E., Roman, G., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+systems+in+Drosophila:+a+synthesis+of+time+and+space.">Gene expression systems in Drosophila: a synthesis of time and space.</a> Trends in Genetics: TIG. 20, 384-391 (2004).</li><li>Smirnova, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Neh2-luciferase+reporter+and+its+application+for+high+throughput+screening+and+real-time+monitoring+of+Nrf2+activators.">Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.</a> Chem. Biol. 18, 752-765 (2011).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-mediated+disruption+of+Wnt-dependent+signaling+in+tissue+regeneration+and+cancer.">Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.</a> Nat. Chem. Biol. 5, 100-107 (2009).</li><li>Tang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+for+Wnt/beta-catenin+pathway+components+identifies+unexpected+roles+for+TCF+transcription+factors+in+cancer.">A genome-wide RNAi screen for Wnt/beta-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9697-9702 (2008).</li><li>Lum, L. D., Kulak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Luciferase+Reporter+Assay+Systems.">Multiplexed Luciferase Reporter Assay Systems.</a> United States patent. , (2012).</li><li>Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E., Shay, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptionally+active+DNA-binding+site+for+human+p53+protein+complexes.">A transcriptionally active DNA-binding site for human p53 protein complexes.</a> Mol. Cell Biol. 12, 2866-2871 (1992).</li><li>DasGupta, R., Kaykas, A., Moon, R. T., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+the+Wnt-wingless+signaling+pathway.">Functional genomic analysis of the Wnt-wingless signaling pathway.</a> Science. 308, 826-833 (2005).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+epithelial+stem-cell+compartments+in+the+small+intestine+of+mice+lacking+Tcf-4.">Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.</a> Nat. Genet. 19, 379-383 (1998).</li><li>Korinek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+members+of+the+Tcf+family+implicated+in+Wnt/beta-catenin+signaling+during+embryogenesis+in+the+mouse.">Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.</a> Mol. Cell Biol. 18, 1248-1256 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487, 330-337 (2012).</li><li>Koboldt, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+portraits+of+human+breast+tumours.">Comprehensive molecular portraits of human breast tumours.</a> Nature. , (2012).</li><li>Wood, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+landscapes+of+human+breast+and+colorectal+cancers.">The genomic landscapes of human breast and colorectal cancers.</a> Science. 318, 1108-1113 (2007).</li><li>Berndt, J. D., Biechele, T. L., Moon, R. T., Major, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+genome-wide+RNA+interference+screens.">Integrative analysis of genome-wide RNA interference screens.</a> Science Signaling. 2, pt4 (2009).</li><li>Falschlehner, C., Steinbrink, S., Erdmann, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+to+dissect+cellular+pathways:+a+how-to+guide.">High-throughput RNAi screening to dissect cellular pathways: a how-to guide.</a> Biotechnology Journal. 5, 368-376 (2010).</li><li>Boutros, M., Bras, L. P., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-based+RNAi+screens.">Analysis of cell-based RNAi screens.</a> Genome Biology. 7, R66 (2006).</li><li>Gilbert, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multiplex+cell+viability+assay+for+high-throughput+RNAi+screening.">A novel multiplex cell viability assay for high-throughput RNAi screening.</a> PLoS ONE. 6, e28338 (2011).</li></ol>

OK와 LL는 루시 페라 기술을 다중화와 관련된 특허 출원에 저자입니다.

처음으로 높은 처리량 화면을 접대하는 데 유용에 대한 온라인 리소스를 제공 루시퍼 기반 기자 시스템의 여러 조달업자 (예 : Promega 사 등, 대상 시스템, 뉴 잉글랜드 Biolabs가, 그리고 써모 피셔)가 있습니다. 또한, 유전자 발견 방법 및 RNAi의 기반 검사 결과는 다른 - 오 믹스 기반 데이터 셋과 통합 될 수 높은 처리량 스크린의 사용을 최적화에 대한 훌륭한 리뷰의 숫자는 도움 15,16해야합니다.<...

세포 생물 학적 과정의 다양한 배열을 모니터링 루시퍼 기반 기자의 다양한 상업적으로 사용할 수 있습니다. 이러한 DNA의 대부분은 특정 세포 자극이나 교란에 의해 표현하는 유도 루시퍼 단백질을 인코딩 구성합니다. 가장 강력한 transcriptionally 기반 기자 합성 증강 요소 또는 유전자의 프로모터 영역 생리학 관련 전사 이벤트 1,2를보고 자신의 신뢰성을 잘 확립의 제어하에 루시 페라 제 효소의 발현을 배치합니다. 루시 페라 제 단백질 발현을 유도하는 GAL4-UAS를 활용 횡단 기자 루시퍼 시스템도 있습니다. GAL4는 효모 전사 활성과 UAS는 GAL4은 특별히 3의 하류에 배치 유전자 서열의 전사를 활성화하기 위해 바인딩을 증강이다. 루시퍼 시스템의 이러한 유형은 일반적으로 두 개의 DNA 플라스미드에 의해 지원됩니다 - 하나는 조절기와 융합 GAL4 단백질을 인코딩스피 관심의 단백질의 부분, 두 번째는 하나 이상의 UAS 시퀀스의 제어 아래에 배치 루시 페라 제 단백질을 인코딩합니다. 따라서, 세포 루시 페라 제 신호가 관심의 단백질의 활동 수준을 반영합니다. 루시 페라 기반 기자의 또 다른 일반적인 사용은 그 단백질 루시 페라 제 효소 4 융합 그것에 의하여 단백질의 안정성을 모니터링합니다. 하나의 잘 심문 한 어떤 기자의 특이성을지지 않습니다 수에 관계없이 기자의 종류,주의 위험 부담이 여기에 기록됩니다. 따라서, 실사가 어떤 연구 전략의 새로​​운 리포터 구조의 결합이 필요합니다. 루시 페라 제 효소 읽기 중 선택의 발현을 제어하는​​ DNA 요​​소의 신뢰성만큼 중요 할 수 있습니다. 반딧불 루시 페라 제 (FL)에 시판 구조에서 가장 일반적으로 사용되는 효소 반면, (경우와 같이 ATP를 필요로하지 않는 새로운 루시 페라 제 리포터 시스템의 출현FL-기반 반응) 또는 그이 연구 플랫폼의 효율성과 안정성을 개선 할 것을 약속 강한 신호를 방출 안정 효소를 통합합니다. 화학 연구에서 루시 페라 제 기자의 선택에 관한 중요 사항을 거짓보고에 상승을 줄 수있는 화학 물질 억제 FL의 감수성이다. 우리의 경험에서, 기판으로 coelenterazine 활용 등 Renilla의 또는 Gaussia 루시 페라 제 (각각 RL 및 GL) 등의 효소가 덜 쉽게 작은 분자 5에 의해 억제합니다. 다중 루시 페라 읽기 아웃에 대한 가장 일반적인 형식은 주로 순차적으로 하나의 샘플에서 효소 활동을 측정 할 수있는 기능을 사용하기 쉬운 키트의 가용성을 부여 FL과 RL의 사용을 수반한다. 대부분의 키트에 샘플을 먼저 동시에 secon를 산출하기 coelenterazine에 기탁되어 담금질 시약 다음 FL 활동의 수준을 나타 내기 위해 루시페린에 노출되는dary RL 신호. 이러한 GL하거나 사용하는 등 Cypridina 루시 페라 제 (CL), luciferases를 사용하여 높은 콘텐츠 데이터를 등장 생성을위한 가능성의 큰 번호와 같은 또 다른 기질로 분비 될 수있는 다른 luciferases의 추가와 함께. 이 기술을 사용하여 게놈 전체의 RNAi 화면의 예는 다음 6 찾을 수 있습니다. 이러한 기술적 진보는 차례가 크로스 토크 7을 제한하기 위해 루시 페라 제 효소의 각 유형을 냉각하는 특정 메커니즘의 개발에 박차를 가했다. 우리 손에, 우리는 GL 또는 CL 등의 분비 luciferases와 &quot;가장자리 효과&quot;(높은 역가 문화 접시의 가장자리와 관련된 세포 활동의 설명 할 수없는 경향)의 큰 주파수를 확인합니다. 그들은 세포 생존을 adulterating없이 신호 샘플링을 가능으로, 다른 한편으로는, 이러한 분비 luciferases은 운동 분석에 유용합니다. 여기에 제시된 연구를 위해, 우리는 동시에 세 개의 암 관련의 활동을 모니터링 할세포 과정 : p53의, 크라스, 그리고 Wnt의 신호 전달 경로. ,, 우리의 연구에 통합 기자 Clontech 사에서 pp53-TA-뤽 FL 기자 (이제부터는 p53의-FL 기자라고도 함) 8 Elk1-GAL4/UAS-CL 기자 시스템 (애질런트 이제부터는 엘크-1 기자)입니다 와 T-세포 인자 (또는 TCFS) 9-11로 알려진 Wnt의 경로 전사 규제에 의해 인식 여러 합성 증강 요소를 통합 8X TCF RL 기자.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

RNA 간섭 (RNAi의)를 사용하여 유전자 기능의 게놈 규모의 심문은 화학적으로 취급하기 쉬운 암 세포 취약점의 신속한 식별을위한 엄청난 약속을 보유하고 있습니다. 이 기술의 잠재력을 제한하면 신속하게 표현형 결과의 기계론의 기초를 묘사함으로써 분자 표적 치료 전략의 개발을 알리는 무능력이다. 우리는 주요 암 세포 관련 프로세스를 모니터링 할 수 있도록 다중 기자 시스템을 사용하여 대상 RNAi를 매개 유전자에 의한 세포의 표현형을 해체하기 위해 여기 방법을 설명합니다. 이 높은 콘텐츠 심사 방법은 다재다능하고 쉽게 큰 분자 라이브러리의 다른 종류의 검사를 위해 적용 할 수 있습니다.

대규모 게놈 시퀀싱 노력 12-14를 사용하여 다양한 암의 돌연변이 풍경을 매핑의 발전에도 불구하고, 우리는 여전히 그 자리에 개입 전략에 이러한 관측을 번역하기위한 체계적인 접근 방법을 가지고 있지 않습니다. / Wnt는 β-catenin 신호, p53의, 그리고 크라스 신호 - - 대장 암 (CRC), 세 가지 세포 프로세스의 구성 요소에 영향을 미칠 것으로 예상되는 돌연변이들은 창자의 세포 변화의 주요 ?...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

세포 프로세스의 시스템 수준의 이해를 달성하는 것은 현대 세포 생물학의 목표입니다. 우리는 여기에서 다양한 세포 기능의 다중 루시 페라 제 기자에 대한 전략을 설명하는 엔드 포인트 게놈 규모의 RNAi 라이브러리를 사용하여 유전자 기능을 심문.

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

a-multiplexed-luciferase-based-screening-platform-for-interrogating

Research

JoVE Journal

Medicine

11.0K 조회수.  UT Southwestern Medical Center. 세포 프로세스의 시스템 수준의 이해를 달성하는 것은 현대 세포 생물학의 목표입니다. 우리는 여기에서 다양한 세포 기능의 다중 루시 페라 제 기자에 대한 전략을 설명하는 엔드 포인트 게놈 규모의 RNAi 라이브러리를 사용하여 유전자 기능을 심문.

비디오: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

지역 Gyrification 인덱스를 계산하기위한 단계별 자습서 : MR 이미지에서 두피 접기를 측정하는 방법

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

연구

의학

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

Neurodegenerative 질병 높은 콘텐츠 심사

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

작은 쥐의 Cystometry의 사용 : 방광 Chemosensation의 연구

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Zebrafish Autochthonous 종양 모델을 사용하여 흑종 조절을위한 검사

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

높은 콘텐츠<em&gt; 체외</em&gt; 췌장 작은 섬의 β 세포 복제 디스커버리 플랫폼

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Toll-like Receptor Signaling의 억제제에 대한 식세포 면역 세포에서의 생체 활성 나노 입자의 스크리닝

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

혈소판 수혈에 미에 대 한 메서드를 검사 하는 루틴

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

히드라, 심장 혈관 분석 및 진단에 임상을 방 조에 대 한 컴퓨터 기반 플랫폼

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

생체 외에서 교양된 폐 포 상피 세포에서 할로겐화 가스의 농도 제어 하는 방법

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...