저자 DRS 감사하고 싶습니다. 릭 라이언, Umesh 파텔, 제프까지, 매튜 수의, Jehangir Mistry 이러한 프로젝트 및 프로토콜의 개발 기간 동안 지원 미쉘 Hatler.

셀 준비 : <ol><li> 성장 미디어 분석, 문화 뉴런 / astrocytes에 대한 셀 시딩 이전까지 ~ 70-80%의 합류 (세포 해동하거나 고립에서 직접 시딩 제외). </li><li> 관심 세포 유형에 적절한 방법을 통해 문화 flasks / 접시에서 세포를 분리합니다. 필요한 경우, 폴리 - D - 라이신 또는 세포외 기질 단백질로 코트 분석 플레이트 우물은 세포 접착력을 향상시키기 위해. astrocytes와 뉴런의 공동 문화가 동시에 또는 순차적으로 씨앗을 품고있을 수 있습니다. 순차 seedings 들어, &quot;기초&quot;레이어 첫번째 astrocytes의 도금이 필요한 경우의 연결을 도금과 차별화 뒤에, 권장합니다. 셀 유형, 후속 문화 시간, 관심의 매개 변수에 맞게 셀 밀도를 조절 등 문화 시대에 따라, 세포의 소스와 시딩 밀도는, 기본 문화 확산, GFAP 표현이나 neurite의 파생물의 속도에 크게 차이가있을 수 있습니다 - 그것은 중요합니다 특징과 가장 생물 학적 귀하의 실험 모델 관련뿐만 아니라 HCS를 (그림 1 참조)를 사용하여 효과적인 영상, 세분화 및 분석을 위해 제공을 제공하기 위해 전지 시스템을 최적화합니다. 플레이트 (셀은 다음 3 단계에서 설명한대로 독소의 치료를 촉진하기 위해, 90 μL 언론에 씨앗을 품고있을 수 있습니다)에 세포를 추가한 후에, 플레이트도 휴대폰 유통을 활성화, 15-30 분 실온에서 수준의 표면에 앉을 수 있습니다. 이 기간에 따라, 적어도 24 시간 동안 성장 미디어에 품어 세포 (37 ° C / 5% CO 2) 다음, 차별화 문화 조건으로 전환 (예를 들어, PC12 세포에 대한 낮은 혈청 / NGF), 적절하게. 세포가 세포 유형에 적절한 간격으로 미디어를 변경 합류 또는 분화의 원하는 수준에 도달할 때까지 문화를 계속합니다. </li><li> 세포 치료 (제어 화합물, 시험 화합물 등)의 관심 치료 시간 코스에 따라,이 문화 기간 동안 어느 시점 도입하실 수 있습니다. 아크릴 아미드, 과산화수소와 K - 252A는 neurotoxic 제어 화합물로 제공됩니다. 충분한 시약은 모두 다섯 96 - 웰 플레이트에 대한 중복 12 포인트 선량 반응 곡선 (선량 반응 내의 DH 2 O 또는 DMSO 제어 세트 포함) 제공됩니다. 화합물은 250X 농도 (K - 252A에 대해 1 μm의 최대 치료를 가정) 또는 10X (아크릴 아미드와 과산화수소에 대해 각각 100 MM 10 MM, 최대 트리 트먼트를 가정)에서 제공합니다. 추천 치료 준비 반 로그인 관련 :에 복잡 희석 버퍼에 희석하여 다음 (1 √ 10) DMSO의 250X 화합물의 시리얼 희석을 10X (DH 2 O에서 발생하는 10X 제어 화합물은 순차적으로 살균 DH 2 O에 직접 희석 수 있습니다 또는 복합 희석 버퍼). 각각의 치료 10 μL 그런 다음 최종 1X 농도 (0.4 % DMSO 또는 10% DH 2 O)에 대한 각 우물에 이미 현재 문화 미디어의 90 μL에 추가할 수 있습니다. 샘플 데이터는 ° C 이전에 고정하기 37 복합 치료의 24 또는 96시간 제공됩니다. </li></ol> 세포 고정과 Immunofluorescent 스테 이닝 : 참고 : 스테 이닝 시간이 ~ 2.5 시간 후 고정합니다. 우물은 얼룩 단계 사이 건조하는 것을 허용하지 않습니다. 시약의 열망과 분배는 유체 전단으로 인한 세포 손실을 감소하기 위해 낮은 흐름 속도에서 실시하여야한다. 모든 권장 볼륨 &#39;잘 당&#39;은 96 잘 microplate 잘 하나를 참조하십시오. 볼륨 &#39;96 - 웰 플레이트마다&#39;전체 권장 액체 처리 볼륨 손실 25 % 초과를 포함합니다. 모든 얼룩 단계 실온 (RT)에서 수행됩니다. 모든 버퍼와 항체 솔루션은 RT 적어도 24 시간 동안 안정합니다. <ol start="4"><li> 문화 기간의 끝에, 실온 (RT) 또는 37 º C로 사전에 따뜻한 HCS의 고정 솔루션 (2X) (12 mL/96-well 플레이트) 원하는 경우. 화학 펌 모자 / 물론 직접적인 문화 매체에 100 μL를 추가하고 RT에서 30 분 수정하실 수 있습니다. (MSDS 참조) 정착액 / 독소 함유 미디어를 제거하고 유해 폐기물에 대한 규정에 따라 처리. 얼룩 즉시 진행하는 경우, HCS Immunofluorescence 버퍼 200 μL와 각각 두 번 잘 씻어. 또는, 접시는 나중에 스테인드 수있다면, 우물과 긴밀하게 얼룩까지 4 º C에서 밀봉 저장 플레이트에서 두 번째 린스 볼륨을두고 다음 씻어 버퍼 200 μL로 두 번 씻어. </li><li> 고정 샘플 전에 얼룩 4 º C에 저장되어있다면, 얼룩 프로토콜하기 전에 200 μL HCS Immunofluorescence 버퍼로 두 번 씻어. </li><li> 다음과 같이 래빗 방지 βIII Tubulin / 마우스 방지 GFAP HCS 차 항체 (6 mL/96-well 판)의 솔루션을 작업 준비 : HCS Immunofluorescence 버퍼의 5.88 ML 각 해동 기본 항체의 60 μL를 추가합니다. 잘 섞는다. 이전 Immunofluorescence 버퍼 린스 제거합니다. 각 잘하는 차 항체 용액 50 μL를 추가하고 RT에서 1 시간 동안 부화. </li><li> 차 항체를 제거솔루션입니다. 200 μL HCS Immunofluorescence 버퍼로 세 번 씻어. </li><li> 다음과 같이 HCS 차 항체 / Hoechst HCS 원자력 스테인 (6 mL/96-well 판)의 작업 솔루션을 준비 : 각 해동 이차 항체의 30 μL 및 해동 Hoechst HCS 원자력의 30 μL HCS Immunofluorescence 버퍼의 5.91 ML에 얼룩을 추가합니다. 빛과 솔루션을 보호, 잘 섞는다. 이전 HCS Immunofluorescence 버퍼가 씻어 제거합니다. HCS 차 항체 / Hoechst HCS 원자력 스테인 솔루션을 50 μL를 추가하고 빛으로부터 보호 RT, 1 시간 동안 부화. </li><li> HCS 차 항체 / Hoechst HCS 원자력 스테인 솔루션을 제거합니다. 200 μL HCS Immunofluorescence 버퍼로 두 번 씻어. </li><li> 이전 HCS Immunofluorescence 버퍼가 씻어 제거합니다. 우물에서 두 번째 린스 볼륨을 떠나, HCS 워시 버퍼 200 μL로 두 번 씻어. </li><li> 4 º C 즉시 인감 판과 이미지 또는 저장 판은 영상에 대한 준비까지 빛으로부터 보호. </li></ol> 이미지 수집 및 분석 : <ol start="12"><li> 스테인드 접시의 이미징 및 분석은 세포 분석기 (GE 헬스케어)를 포​​함 가능 HCS 플랫폼, ArrayScan (ThermoFisher 과학) 또는 오페라 (퍼어킨 엘머)의 다양한시 수행할 수 있습니다. 이미징 및 분석을위한 몇 가지 지침은 표 1에서 제공됩니다 : </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222 이미지 수집 가이드 </td></tr><tr><td> 탐지 시약 </td><td> 목표 렌즈 </td><td> 여기 필터 범위 [피크 / 대역폭 (NM)] </td><td> 배출 필터 범위 [피크 / 대역폭 (NM)] </td></tr><tr><td> Hoechst HCS 핵은 스테인 </td><td> 20X </td><td> 40분의 360 </td><td> 40분의 460 (또는 필요한 경우 50분의 535) </td></tr><tr><td> HCS 차 항체, FITC - 돈키 안티 - 토끼 IgG </td><td> 20X </td><td> 40분의 480 </td><td> 50분의 535 </td></tr><tr><td> HCS 차 항체, Cy3 - 돈키 안티 - 마우스 IgG </td><td> 20X </td><td> 50분의 535 </td><td> 50분의 600 </td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222 이미지 분석 가이드 </td></tr><tr><td> 세포 매개 변수 </td><td> 검색 </td><td> 세그먼트 / 측정 </td><td> 이론적 해석 </td></tr><tr><td> 휴대폰 번호, 핵 특성 </td><td> Hoechst HCS 핵은 스테인 </td><td> 원자력 지역 (460 NM 방출 채널). 핵의 개수를 계산합니다. DNA 내용 (핵 강도) 또는 핵 영역 분석도 가능합니다. </td><td> 별도의 연결 및 astrocytic 셀 카운트 / characterizations를 (권장)을 구하는 βIII - tubulin 또는 GFAP 표현과 관련된들을 위해 핵을 &quot;필터링&quot;할 수 있습니다 셀 손실, 독성 phenotypes 등을 확인하기 위해 휴대폰 번호, 핵 특성을 사용합니다. </td></tr><tr><td> βIII - Tubulin의 표현, Neurite의 파생물 </td><td> HCS 차 항체, FITC - 복합 </td><td> 세포질 영역 (535 nm의 방출 채널). FITC 신호 neurites (예 : 최소 / 평균 셀 신체 영역, 최소 / 최대 neurite의 길이와 넓이를 통해)에서 세포의 연결 기관을 구분하는 데 사용할 수 있습니다. 등 총 neurite의 길이, neurite 카운트 / 휴대폰 등 매개 변수를 결정 </td><td> Neurite의 가지 측정의 연결을 차별화하는 동안이나 화학 부상, 질병 상태 등의 결과로 변조된 수 있습니다 별개의 연결 특성을 (권장)을 구하는 βIII - tubulin 표현과 관련된들을위한 &quot;필터&quot;셀 단체 수있다. </td></tr><tr><td> GFAP의 표현, Astrocyte 지역 </td><td> HCS 차 항체, Cy3 - 복합 </td><td> 세포질 영역 (600 NM 방출 채널). Cy3 신호 astrocytic 세포질 세그먼트를 정의하는 데 사용할 수 있습니다. 이러한 평균 세포질 신호 강도, 셀 영역 등 매개 변수를 결정 </td><td> GFAP의 표현과 astrocyte 세포 영역 astrocyte 개발하는 동안이나 화학 부상, 질병 상태의 결과로 변조된 수 등 독특한 astrocytic 특성화를 (권장)을 구하는 GFAP 표현과 관련된들을위한 세포 기관을 &quot;필터링&quot;할 수도있다. </td></tr></tbody></table> . 표 1 이미지 수집 및 분석 지침 - HCS222 βIII-Tubulin/GFAP (CO - 문화) 어세이 대표 결과 : <img src="/files/ftp_upload/1173/figure31173.png" alt="그림 3" /> 그림 1. βIII - tubulin과 GFAP immunofluore혼합 쥐의 신경 세포 문화의 카메라는. 기본 쥐 hippocampal 뉴런 또는 쥐 PC12 세포 중 하나와 함께 기본 쥐 hippocampal astrocytes의 공동 문화가의 연결 시딩 다음에 문화에 며칠에 대한 사전 도금 astrocytes에 의해 생성되었습니다. PC12s가 차별화의 조건 (낮은 혈청 / NGF)에서 추가로 2 일간 교양 동안 차 뉴런은 추가 11일에 대한 교양되었습니다. 세포 처리, 고정 및 immunostaining은 HCS222 분석 프로토콜에 따라 수행되었다. 전지는 20X (주 뉴런) 또는 10X (PC12) 객관적인 배율에서 세포 분석기 1000 (3.3)에서 GE에 몇 군데와 세포 분석기 1000 워크 스테이션 (3.4) Neurite의 파생물 및 멀티 타겟 분석 알고리즘에서 GE를 사용하여 분석되었다 톱 패널. : Hoechst HCS 스테인 원자력 (파란색), βIII - tubulin (녹색) 및 GFAP (적색) 형광의 융합 영상. (세포 기관 파란색 (주 뉴런에 명시된) 또는 노란색 (PC12), 그린 (주 뉴런) 또는 하늘색 (PC12)에 neurites 가운데 패널) βIII - tubulin의 형광 채널의 분리 분석 neurite 가지 세분화를위한 수 있습니다. GFAP 채널의 분석 astrocyte 세분화 (핵 녹색 파랑, 세포질에 명시된 하단 패널)에 있습니다. 에 대해 별도의 셀 계산을 지원, (적색) (-) 기본 쥐 hippocampal 신경 / astrocyte 공동 문화 GFAP 세분화는 핵 / 세포 GFAP (+) (녹색 윤곽선)입니다 신체와 GFAP하는 사람을 구별하는 능력을 보여줍니다 혼합 문화 환경에서 뉴런과 astrocytes. <img src="/files/ftp_upload/1173/figure41173.png" alt="그림 4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="figure4 파트" /> 그림 2. 기본 쥐 선량 반응 hippocampal 신경 / 독성 스트레스에 공동 문화 astrocyte. 기본 쥐 hippocampal astrocytes는 (P6) 성장 미디어 96 - 웰 플레이트에 도금 6 일 동안 교양되었습니다. 기본 쥐 hippocampal 뉴런은 (해동 직접) 다음 사전 도금 astrocytes 위에 씨앗을 11 일 동안 성장 미디어에 교양되었습니다. 세포 문화의 마지막 24 시간 동안 아크릴 아미드 또는 수소 과산화물 (최대 농도 = 100mM 및 10mM, 각각)의 시리얼 dilutions로 치료했다. 세포 처리, 고정 및 immunostaining은 HCS222 분석 프로토콜에 따라 수행되었다. 전지는 20X (물론 10 필드 /)에서 세포 분석기 1000 (3.3)에서 GE에 몇 군데와 세포 분석기 1000 워크 스테이션 (3.4) Neurite의 파생물 및 멀티 타겟 분석 알고리즘에서 GE를 사용하여 (세포질 / 핵 / neurite 세분화) 분석했다. 표시 데이터 굴어요 ± SEM, N = 3. 여러 매개 변수 (neurite 가지, GFAP 강도, astrocyte 지역의 연결 또는 astrocytic 셀 카운트) 선량에 의존 동향에 대해 조사했다.

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	<li>Radio, N. M., Mundy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neurotoxicity+testing+in+vitro:+models+for+assessing+chemical+effects+on+neurite+outgrowth.">Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth.</a> Neurotoxicology. 29, 361-376 (2008).</li><li>Richards, G. R., Smith, A. J., Parry, F., Platts, A., Chan, G. K., Leveridge, M., Kerby, J. E., Simpson, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphology-+and+kinetics-based+cascade+for+human+neural+cell+high+content+screening.">A morphology- and kinetics-based cascade for human neural cell high content screening.</a> Assay Drug Dev Technol. 4, 143-152 (2006).</li><li>Evans, N. A., Facci, L., Owen, D. E., Soden, P. E., Burbidge, S. A., Prinjha, R. K., Richardson, J. C., Skaper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta(1-42)+reduces+synapse+number+and+inhibits+neurite+outgrowth+in+primary+cortical+and+hippocampal+neurons:+A+quantitative+analysis.">Abeta(1-42) reduces synapse number and inhibits neurite outgrowth in primary cortical and hippocampal neurons: A quantitative analysis.</a> J Neurosci Methods. 175, 96-103 (2008).</li><li>Breier, J. M., Radio, N. M., Mundy, W. R., Shafer, T. J. <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+a+high-throughput+screening+assay+for+chemical+effects+on+proliferation+and+viability+of+immortalized+human+neural+progenitor+cells.">Development of a high-throughput screening assay for chemical effects on proliferation and viability of immortalized human neural progenitor cells.</a> Toxicol Sci. 105, 119-133 (2008).</li><li>Radio, N. M., Breier, J. M., Shafer, T. J., Mundy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+chemical+effects+on+neurite+outgrowth+in+PC12+cells+using+high+content+screening.">Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening.</a> Toxicol Sci. 105, 106-118 (2008).</li><li>Harry, G. J., Tiffany-Castiglioni, E. <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+neurotoxic+potential+by+use+of+in+vitro+systems.">Evaluation of neurotoxic potential by use of in vitro systems.</a> Expert Opin Drug Metab Toxicol. 1, 701-713 (2005).</li><li>Prockop, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+allegory+of+a+mountain:+an+environmental+introduction+to+neurotoxicology.">The allegory of a mountain: an environmental introduction to neurotoxicology.</a> J Neurol Sci. 262, 7-14 (2007).</li><li>Woehrling, E. K., Hill, E. J., Coleman, M. D. <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+a+neurotoxicity+test-system,+using+human+post-mitotic,+astrocytic+and+neuronal+cell+lines+in+co-culture.">Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture.</a> Toxicol In Vitro. 21, 1241-1246 (2007).</li><li>Holden, L. J., Coleman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+astrogliotic+responses+of+three+human+astrocytoma+cell+lines+to+ethanol,+trimethyltin+chloride+and+acrylamide.">Assessment of the astrogliotic responses of three human astrocytoma cell lines to ethanol, trimethyltin chloride and acrylamide.</a> Toxicology. 241, 75-83 (2007).</li><li>R&#246;hl, C., Lucius, R., Sievers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+activated+microglia+on+astrogliosis+parameters+in+astrocyte+cultures.">The effect of activated microglia on astrogliosis parameters in astrocyte cultures.</a> Brain Res. 1129, 43-52 (2007).</li><li>Eng, L. F., Ghirnikar, R. S., Lee, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+fibrillary+acidic+protein:+GFAP-thirty-one+years+(1969-2000).">Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000).</a> Neurochem Res. 25, 1439-1451 (2000).</li><li>Gadea, A., Schinelli, S., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-1+regulates+astrocyte+proliferation+and+reactive+gliosis+via+a+JNK/c-Jun+signaling+pathway.">Endothelin-1 regulates astrocyte proliferation and reactive gliosis via a JNK/c-Jun signaling pathway.</a> J Neurosci. 28, 2394-2408 (2008).</li><li>O'Callaghan, J. P., Sriram, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+fibrillary+acidic+protein+and+related+glial+proteins+as+biomarkers+of+neurotoxicity.">Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity.</a> Expert Opin Drug Saf. 4, 433-442 (2005).</li><li>Mead, C., Pentreath, V. W. <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+toxicity+indicators+in+rat+primary+astrocytes,+C6+glioma+and+human+1321N1+astrocytoma+cells:+can+gliotoxicity+be+distinguished+from+cytotoxicity?.">Evaluation of toxicity indicators in rat primary astrocytes, C6 glioma and human 1321N1 astrocytoma cells: can gliotoxicity be distinguished from cytotoxicity?.</a> Arch Toxicol. 72, 372-380 (1998).</li><li>Balls, M., Walum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11:+towards+the+acceptance+of+in+vitro+neurotoxicity+tests.">Chapter 11: towards the acceptance of in vitro neurotoxicity tests.</a> Neurotoxicology In Vitro. , 269-283 (1999).</li><li>Yokosuka, M., Ohtani-Kaneko, R., Yamashita, K., Muraoka, D., Kuroda, Y., Watanabe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+and+environmental+estrogenic+chemicals+exert+developmental+effects+on+rat+hypothalamic+neurons+and+glias.">Estrogen and environmental estrogenic chemicals exert developmental effects on rat hypothalamic neurons and glias.</a> Toxicol In Vitro. 22, 1-9 (2008).</li><li>Weible, M. W., Chan-Ling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+neural+stem+cells+from+human+fetal+spinal+cord:+synergistic+effect+of+LIF+and+BMP4+to+generate+astrocytes.">Phenotypic characterization of neural stem cells from human fetal spinal cord: synergistic effect of LIF and BMP4 to generate astrocytes.</a> Glia. 55, 1156-1168 (2007).</li><li>Casulari, L. A., Melcangi, R. C., Piva, F., Martini, L., Maggi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+released+by+rat+type+1+astrocytes+exert+different+effects+on+the+proliferation+of+human+neuroblastoma+cells+(SH-SY5Y)+in+vitro.">Factors released by rat type 1 astrocytes exert different effects on the proliferation of human neuroblastoma cells (SH-SY5Y) in vitro.</a> Endocr Relat Cancer. 7, 63-71 (2000).</li><li>Dringen, R., Gutterer, J. 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M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

저자는 Millipore Corporation의 모든 임직원 수 있습니다.

지금까지, 뉴런과 astrocytes의 혼합 문화를 사용하여 neurotoxicity 위험 평가를 공부하기위한 체외 실험에서 제한되었습니다. 그것은 glial 세포가 뉴런에 공간과 신진 대사 지원을 제공 통합하고 있으며,의 연결을 마이 그 레이션, 분화와 시냅스 활동을 포함한 여러 modulating의 연결 기능에 중요한 역할을 것을 잘 허용됩니다. Glial astrocytes 또한 크린 시토킨 많은 독소의 양쪽의 연결 조직을 둘러싼에 불리한 반응을 유도뿐만 아니라 홍보의 연결을 허용 수있는 다른 가용성 요소를 놓습니다. 이러한 항산화 방어하고 세포의 에너지 수준의 유지 보수와 같은 astrocytic 기능의 장애가로 표시되었습니다 반면에의 연결 - glial 상호 작용의 깊이를 보여주는, 공동 문화 연구에 astrocytes은 산화 스트레스의 독성에 대한 뉴런을 보호하기 위해 표시되었습니다 비판적으로의 연결을 생존에 영향을 미칩니다. 최근 연구는 체외의 neurotoxicity 테스트 - 시스템에 astrocytes의 사용 혼자의 연결을 세포보다 인간의 중추 신경계의 구조와 기능 관련성이 될 수 있습니다 것을 제안했습니다. 의 연결 - astrocytic 상호 작용의 생리적 의의의 성장 인식에도 불구하고, 그것은 이전 그대로 세포에서 이러한 상호 작용의 양적 분석을 수행하기 어려운되었습니다. 하이 컨텐츠 스크리닝의 출현은 강력한 새 assays의 개발이이 문제를 해결할 수 있습니다. 본 연구에서는, 우리는 하나의 분석에서 여러의 연결 및 astrocytic phenotypes의 양적 분석은 HCA에 의해 가능하게 한 것을 증명하고있다. 기본 뉴런에서 우리는의 연결 번호, neurite 카운트와 neurite 길이로 neurotoxicity 마커의 양적 측정을 수행했습니다. astrocytes에 반응 gliosis는 GFAP 표현, astrocyte 번호 ​​astrocyte 형태의 변경에 의해 특징 neurotoxicity의 알려진 biomarker입니다. 우리는이 끝점 각각이 쉽게 HCA를 통해 평가 수 증명하고있다. 이 연구는 신경 특정 생체 HCA가 급속하게 neurotoxic 종점에 대한 화학 물질의 큰 숫자를 화면으로 체외 테스트에의 배터리의 일환으로 사용될 수있는 개념을 지원합니다. 뉴런과 astrocytes의 공동 문화 Millipore의 HCS222 하이 컨텐츠 분석 키트는 고품질 탐지 시약 동시에 neurotoxicity의 세포 모델의 다양한 βIII - tubulin과 glial fibrillary 산성 단백질 (GFAP)를 프로 파일링을위한 프로토콜로 구성되어 있습니다. 이 키트와 함께 제공되는 높은 유효 시약은 사용자가 assays을 표준화 분석 - 투 - 분석 변화를 최소화하고, reproducibly 높은 신호 대 배경 비율로 이미지를 생성하실 수 있습니다.

<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rabbit Anti-&#x03B2;III-Tubulin HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201672</td>
<td>1 vial containing 300 &micro;L 
Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201649</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201671</td>
<td>1 vial containing 300 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200437</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hoechst HCS Nuclear Stain, 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200438</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Fixation Solution with Phenol Red, 2X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200434</td>
<td>1 bottle containing 100 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Immunofluorescence Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200435</td>
<td>1 bottle containing 1000 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Wash Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. 2007643</td>
<td>1 bottle containing 500 mL Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>Acrylamide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201679</td>
<td>1 vial containing 500 &micro;L of 1M acrylamide in dH2O Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hydrogen Peroxide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201730</td>
<td>1 vial containing 500 &micro;L of 100 mM hydrogen peroxide in dH2O Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>K-252a (Control Compound), 250X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201741</td>
<td>1 vial containing 100 &micro;L of 250 &micro;M K-252a in DMSO Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>DMSO for Compound Serial Dilution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200441</td>
<td>1 bottle containing 10 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Compound Dilution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200442</td>
<td>1 bottle containing 25 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Plate Sealers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200443</td>
<td>10 each Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>tissue culture-treated black/clear bottom microplates </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HCS imaging/analysis system</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

하이 컨텐츠 분석 (HCA) assays은 단일 분석에서 여러 세포 phenotypes의 측정을 수 있도록 자동 이미징과 강력한 이미지 분석 알고리즘 세포 검출 시약을 결합. 본 연구에서는, 우리는 neurotoxicity에 대한 소설 분석을 개발하는 HCA를 활용. Neurotoxicity 평가는 중요한 약물 안전 평가의 일부뿐만 아니라, 환경 보호 노력의 중요한 초점이되고를 나타냅니다. 또한, neurotoxicity는 잘 접수<em&gt; 체외에서</em등 알츠하이머와 파킨슨병의 질병으로 neurodegenerative 질병의 개발&gt; 마커. 최근의 연결을 검사하기 위해 HCA의 신청이보고되었습니다. βIII - tubulin로의 연결을 세포를 분류하여, HCA의 assays은의 연결 번호, neurite 개수 및 neurotoxic 효과를 나타낼 수있는 모든있는 neurite의 길이로 매개 변수의 높은 처리량이 아닌 주관적인, 양적 측정을 제공할 수 있습니다. 그러나, astrocytes의 역할은이 모델에서 미개척 남아 있습니다. Astrocytes는 중추 신경계 (CNS) 항상성의 유지에 중요한 역할을 가지고 있고, 그들이 독성 물질 또는 질병 상태에 대한 응답으로 활성화됩니다 neuroprotection 및 neurodegradation 모두와 관련됩니다. GFAP는 CNS의 astrocytes에 주로 표현 중간 필라멘트 단백질이다. Astrocytic 활성화 (gliosis)은 일반적으로 증식과 비대 astrocyte 함께 GFAP의 upregulation에 연결됩니다. 반응 gliosis의이 과정은 신경계 손상의 초기 마커로 제안되었습니다. GFAP의 quantitation에 대한 전통적인 방법은 면역입니다. 이러한 접근 방식은 셀룰러 현지화, 형태학 및 휴대폰 번호에 대한 정보를 제공하는 무능력에 의해 제한됩니다. 우리는 HCA 이러한 한계를 극복하기 위해 동시에 gliosis와 관련된 여러 기능을 측정하는 데 사용될 수 있다고 결정 - GFAP 표현의 변화, astrocyte 비대하고, astrocyte 확산 - 단일 분석 이내에 있습니다. 공동 문화 연구에서, astrocytes는 독성 모욕 여러 종류에 대해 신경을 보호하고 비판적으로의 연결을 생존에 영향을 보여왔다. 최근 연구 제안했다고에서 astrocytes의 사용<em&gt; 체외에서</em&gt; neurotoxicity 테스트 시스템은 혼자의 연결을 세포보다 인간 CNS 구조와 기능 관련성이 될 수 있습니다. βIII - tubulin과 glial fibrillary 산성 단백질 (GFAP)를 프로 파일링을위한 따라서, 우리는, 뉴런과 astrocytes의 공동 문화 HCA 분석을 개발 프로토콜의 구성과 검증 있고, 타겟 특정 검색 시약. 이 분석은 neurotoxicity 평가가 아닌 주관적인, 높은 처리량 분석 소설을 대표하는 neurotoxicity의 동시 분석, neurite의 파생물, gliosis, 세포 모델의 다양한의 연결 및 astrocytic 형태학 및의 연결 및 astrocytic 개발을 수 있습니다. 분석은 신경 과학 연구 및 약물 발견에 neurotoxicity와 생산성 향상의 향상된 검출을위한 잠재력을 보유하고 있습니다.

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

이 문서는 소설 프로토콜 및 시약 높은 컨텐츠 분석을 사용하여 뉴런과 astrocytes의 공동 문화 화합물과 치료의 neurotoxic 효과 민감한 측정을위한 설정에 대해 설명합니다. 결과는 높은 컨텐츠 분석 neurotoxicity 평가에 대한 흥미 진진한 소설 기술을 대표하는 것을 보여줍니다.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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JoVE Journal

Biology

25.0K 조회수.  Millipore Inc.  日本語 

비디오: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

성장 콘스의 고해상도 이미징을위한 Aplysia에서의 연결을 셀 문화

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

연구

생물학

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

다중 구획 CNS 뉴런 - glia 공동 문화 Microfluidic 플랫폼

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidic 가용성식이 중재 신호의 상호 작용을 조사하는 상피 세포 박테리아의 공동 문화

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

히타치 F - 7000 형광 분광 및 PicoGreen 염료를 사용하여 dsDNA의 부량

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

파우더 샘플의 절대 양자 수율 측정

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

표현 복제가 Grb2 신호 복합 단지를 공부하기 위해 높은 콘텐츠 이미징 워크 플로우

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

펩티드 에피토프의 정량 분석​​을위한 높은 처리량 MHC II 결합 분석

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...