Name: assaying dna damage in hippocampal neurons using the comet assay
Uploaded: 2012-12-19T13:00:00.000+00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

이 작품은 방사선 종양학, 앨라배마 - 버밍햄 대학 종합 암 센터, 파이팅 어린이 암 재단과 가브리엘의 엔젤 재단 (ESY 있습니다.)의 부에서 IMPACT 상에 의해 지원되었다.

1. 세포 배양 <ol><li> 문화 neuronal 세포와이를 필요에 따라 취급합니다. </li><li> 15 ML 튜브에 수확 세포 : 기음 미디어, 인산염과 린스 (PBS, 칼슘, 마그네슘 무료) 생리 버퍼, 15 ML 튜브에 수집하고 적절한 혈청이 포함 된 미디어와 트립신을 중화, 트립신을 추가합니다. </li><li> 5 분 1,000 XG에서 봐. </li><li> 미디어를 대기음. </li><li> PBS에 세포를 Resuspend. </li><li> 5 분 1,000 XG에서 봐. </li><li> 신선한 PBS에서 미디어 및 resuspend 세포를 대기음. </li><li> hemacytometer이나 선호하는 셀 카운터를 사용하여 셀을 계산합니다. </li><li> 세포 샘플은 분석을 시작하기 바로 전에 준비해야합니다. </li><li> 모든 샘플은 자외선에서 DNA 손상을 방지하기 위해 어둡거나 노란 빛으로 처리해야합니다. </li></ol> 2. 혜성 분석 1. 슬라이드 준비 <ol><li> 1% 아가로 오스를 (1X 트리스베이스, 붕산, EDTA (에틸렌 다이아 민 테트라 초산), TB의 1g / 100 ML 녹음E) 3 분의 전자 레인지의 모든 과립가 사라 때까지. </li><li> 수영 용융 아가로 오스에 슬라이드과 측면을 닦아은 kimwipe으로 청소하십시오. 투명 필름에 공기 건조에 아가로 오스 할 수 있습니다. 이것은 저장할 수 있습니다 사전 및 슬라이드에 수행 할 수 있습니다. </li></ol> 2. 중성 혜성 분석 <ol><li> 사용하기 전에 적어도 1 시간에 ° C 4에 얼음 용해 용액 (2.5 M NaCl, 100 MM EDTA (에틸렌 다이아 민 테트라 초산) 산도 10, 10 MM 트리스베이스, 1 %의 나트륨 라 우릴의 sarcosinate, 그리고 1퍼센트 튼 X-100, 산도 10). </li><li> 2.2.2. 모든 과립이 사라 때까지 전자 레인지에 1% 낮은 녹는 점 아가로 오스는 (1X 트리스 자료의 1g / 100ml, 붕소의 산성, EDTA (에틸렌 다이아 민 테트라 초산)) 3 분 동안 녹음. 아가로 오스는 혜성 꼬리의 인공 유도를 방지하기 위해 ° C 물 목욕에서 37로 냉각해야합니다. 이상적으로, 아가로 오스는 사용하기 전에 반 시간 동안 냉각해야합니다. </li><li> 2.2.3. ML 100,000 세포가되도록 세포 현탁액을 희석. 낮은 녹는 점 아가로 오스 (37시 ° C)에있는 세포 현탁액을 결합1시 10분 (V / V), 와류 짧게의 비율과 즉시 혜성 슬라이드에 50 μl를 피펫. 샘플 영역 골고루 세포 현탁액을 확산하기 위해 피펫 팁의 측면을 사용합니다. 각 치료 그룹은 적어도 세중의에 있어야합니다. </li><li> 원은 슬라이드의 주변에 나타날 때까지 장소 30 분에 냉장고에 평면 슬라이드. </li><li> Prechill 4에 어둠 속에서 30 분이 솔루션에 용해 솔루션과 잠수함 슬라이드 ° C (또는 냉장고에). </li><li> 오프 넣어 또는 용해 솔루션을 기음과 1X 중립적 인 전기 영동 버퍼 (트리스베이스, 붕산, EDTA (에틸렌 다이아 민 테트라 초산), 1X TBE)을 추가합니다. 냉장고에 30 분 동안이 버퍼에 슬라이드를 둡니다. </li><li> 전기 챔버에서 prechilled 1X 중성 전기 영동 버퍼 (TBE), 전기 슬라이드 트레이에 장소 슬라이드를 추가 할 수 있습니다. 사람들이 전극에서 등거리되도록 슬라이드를 맞 춥니 다. </li><li> 1X 중립 전기가 슬라이드 위에 0.2 인치까지 버퍼 네요. 초과 버퍼 간 것전기와 fere. </li><li> cm 당 1 V (전극에 전극을 측정)에 전원 공급 장치의 전압을 설정하고 어둠 속에서 4 ° C (또는 차가운 방)에서 30 분에 실행합니다. </li></ol> 3. 알칼리성 혜성 분석 <ol><li> 사용하기 전에 적어도 1 시간에 ° C 4에 얼음 용해 용액 (2.5 M NaCl, 100 MM EDTA (에틸렌 다이아 민 테트라 초산) 산도 10, 10 MM 트리스베이스, 1 %의 나트륨 라 우릴의 sarcosinate, 그리고 1퍼센트 튼 X-100, 산도 10). </li><li> 모든 과립이 사라 때까지 3 분을 위해 전자 레인지에 1% 낮은 녹는 점 아가로 오스를 (1X 트리스 자료의 1g / 100 ML, 붕소의 산성, EDTA (에틸렌 다이아 민 테트라 초산)) 녹음. 그럼 적어도 30 분에 37 ° C의 물을 욕조에 좋아. </li><li> 1시 10분 (V / V), 와류 잠시의 비율로 용융 낮은 녹는 점 아가로 오스와 1 개 105 / ML (37시 ° C)에서 세포를 결합하고 즉시 혜성 슬라이드에 50 μl를 피펫. 샘플 영역 골고루 세포 현탁액을 확산하기 위해 피펫 팁의 측면을 사용합니다. 각 치료 그룹은 적어도 세중의에 있어야합니다. </li><li> 장소는 미끄러에스 평면은 30 분에 냉장고에있는 원은 슬라이드의 주변에 나타납니다 때까지. </li><li> prechilled 용해 용액과 4에 남겨 ° C를 어둠 속에서 야간에 1 시간에 젖어 슬라이드. </li><li> 용해 용액에서 슬라이드를 제거 슬라이드를 배출하고 알칼리 긴장을 풀기 단계로 사전에 잔류 세제와 소금을 제거하는 5 분을위한 차가운 중화 버퍼로 한 번 씻어. </li><li> 겔 전기 영동 챔버의 장소 슬라이드 슬라이드 위에 0.5 cm를 초과하지 prechilled 갓 만든 전기 영동 버퍼 (300 MM NaOH, 1 MM EDTA (에틸렌 다이아 민 테트라 초산),&gt; 13 PH)로 가득. 사람들이 전극에서 등거리되도록 슬라이드를 맞 춥니 다. </li><li> 슬라이드가 DNA와 알칼리 - 책임 손상의 표현의 긴장을 풀기을 할 수 있도록 어둠 속에서 30 분 동안 알칼리 버퍼에 앉아 보자. </li><li> cm 당 1 V (전극에 전극을 측정)에 전원 공급 장치의 전압을 설정하고 4시 30 분에 실행 ° C (또는 추운 방). </li></ol> 4. 고정과 역ining 셀 <ol><li> 초과 전기 버퍼를 배수 </li><li> 젖어은 RT에서 5 분을 위해 사전에 차가운 증류수에 슬라이드. </li><li> RT에서 5 분을위한 사전 차가운 70 % 에탄올에 젖어 슬라이드. </li><li> 밤새 드라이 샘플. 밝은 빛에 슬라이드를 노출시키지 마십시오. 샘플은 이전 단계에서 골을 실온에서 몇 달 동안 저장 될 수 있습니다. </li><li> 냉장고에 20 분에 Sybr 녹색 (PBS에 희석 1X)에 immersing하여 슬라이드를 청바지. </li><li> 슬라이드를 제거하고 그 어둠 속으로 완전히 건조 할 수 있습니다. 때 완전히 건조 아가로 오스는 투명이 될 것입니다. </li></ol> 4. 이미지 수집 및 분석 <ol><li> 녹색 필터 (Zeiss AxioVision)와 혜성 분석 소프트웨어 (지각 악기)를 사용하여 분석으로 설정 형광 현미경을 사용하여 이미지를 획득. </li><li> &quot;혜성의 머리&quot;(핵)를 클릭하고 소프트웨어에서 평균 꼬리 순간 DNA의 양을 포함하여 매개 변수를 계산핵. </li><li> 치료 당 최소한 200 세포를 분석합니다. </li><li> Microsoft Excel로 데이터를 내보낼 수도 있습니다. </li><li> 각 치료 그룹과 적절한 플롯 데이터 꼬리 순간을 의미 계산합니다. </li></ol> 5. 대표 결과 neuronal 세포에 혜성 분석 분석의 예는 그림 2와 그림 3에 표시됩니다. 이 경우 neuronal 세포의 방사선은 DNA 손상을 유도한다. 세포는 전기 분야의 대상이됨에 따라, DNA는 이후 혜성 분석 소프트웨어를 사용하여 분석 크기의 차이로 인해 서로 다른 속도로 마이그레이션합니다. 더 DNA 손상은 멀리 DNA는 핵에서 마이그레이션합니다. 이 이후 높은 꼬리 순간의 소프트웨어와 결과에 의해 포착 된 핵의 형광 강도를 낮 춥니 다. 표 1은 혜성 분석 분석 및 그림 4에서 대표 표를 묘사 represen를 보여줍니다tative 그래프는 혜성 분석에 의해 측정으로 방사선에 따라 neuronal 세포의 DNA 손상을 비교. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="그림 1"/> 그림 1. 혜성 분석의 흐름 차트. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="그림 2"/> 그림 2. neuronal 세포의 대표 이미지 (A)없이와 (B) 혜성 꼬리. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="그림 3"/> 혜성 분석 소프트웨어를 사용하여 그림 3. 혜성 분석 분석. (A) 이미지의 대표 스크린 샷은 칼 Zeiss 형광 현미경을 사용하여 획득과 혜성을 사용하여 분석분석 소프트웨어. (B) 핵 또는 &quot;혜성의 머리&quot;(녹색 동그라미)을 클릭하면 형광지도와 그래프 (노란색 동그라미)와 (C) 데이터 테이블을 (빨간색 원)으로 생성합니다. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="그림 4"/> 그림 4. 중립적 인 혜성 분석을 사용하여 방사능과 비 방사능 neuronal 세포의 DNA 손상을 분석하여 얻은 평균 꼬리 순간을 음모하여 얻은 대표 그래프. 예상대로 방사능 neuronal 세포에서 높은 평균 꼬리의 순간에 의해 묘사로 3Gy 방사선 (x-선)은 DNA 손상을 유도한다. , ** P &lt;0.01 - 평균 꼬리 순간 (표준 오류 + /)입니다 표시됩니다. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="표 1"/> 표 1. </STRONG&gt; 방사능과 비 방사능 neuronal 세포에 혜성 분석 분석 얻은 대표 표. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> .

<ol>
	<li>Olive, P. L., Banath, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+a+method+to+measure+DNA+damage+in+individual+cells.">The comet assay: a method to measure DNA damage in individual cells.</a> Nat. Protoc. 1, 23-29 (2006).</li><li>Hovhannisyan, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+in+combination+with+the+comet+assay+and+micronucleus+test+in+genetic+toxicology.">Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology.</a> Mol. Cytogenet. 3, (2010).</li><li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay.+An+overview+of+techniques.">The comet assay. An overview of techniques.</a> Methods Mol. Biol. 203, 179-194 (2002).</li><li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+DNA+damage+in+individual+cells+by+analysis+of+histone+H2AX+phosphorylation.">Detection of DNA damage in individual cells by analysis of histone H2AX phosphorylation.</a> Methods Cell Biol. 75, 355-373 (2004).</li><li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+comet+assay+in+radiobiology.">Impact of the comet assay in radiobiology.</a> Mutat. Res. 681, 13-23 (2009).</li><li>Olive, P. L., Banath, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+H2AX+phosphorylation+after+exposure+to+cisplatin.">Kinetics of H2AX phosphorylation after exposure to cisplatin.</a> Cytometry B Clin. Cytom. 76, 79-90 (2009).</li><li>Olive, P. L., Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+DNA+damage+using+the+comet+assay.">Heterogeneity in DNA damage using the comet assay.</a> Cytometry A. 66, 1-8 (2005).</li><li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+for+DNA+damage+and+repair.">The comet assay for DNA damage and repair.</a> Molecular biotechnology. 26, 249-261 (2004).</li><li>Fairbairn, D. W., Olive, P. L., O'Neill, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+a+comprehensive+review.">The comet assay: a comprehensive review.</a> Mutation Research/Reviews in Genetic Toxicology. 339, 37-59 (1995).</li><li>Yang, E. S., Nowsheen, S., Wang, T., Thotala, D. K., Xia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+synthase+kinase+3B+inhibition+enhances+repair+of+DNA+double-strand+breaks+in+irradiated+hippocampal+neurons.">Glycogen synthase kinase 3B inhibition enhances repair of DNA double-strand breaks in irradiated hippocampal neurons.</a> Neuro-Oncology. 13, 459-470 (2011).</li><li>Yang, E. S., 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=Lithium-mediated+protection+of+hippocampal+cells+involves+enhancement+of+DNA-PK+dependent+repair+in+mice.">Lithium-mediated protection of hippocampal cells involves enhancement of DNA-PK dependent repair in mice.</a> The Journal of clinical investigation. , 119-1124 (2009).</li><li>Collins, A. R., Harrington, V., Drew, J., Melvin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+modulation+of+DNA+repair+in+a+human+intervention+study.">Nutritional modulation of DNA repair in a human intervention study.</a> Carcinogenesis. 24, 511-515 (2003).</li><li>Smith, C. C., O'Donovan, M. R., Martin, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hOGG1+recognizes+oxidative+damage+using+the+comet+assay+with+greater+specificity+than+FPG+or+ENDOIII.">hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII.</a> Mutagenesis. 21, 185-190 (2006).</li><li>Rosenquist, T. A., Zharkov, D. O., Grollman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+a+mammalian+8-oxoguanine+DNA+glycosylase.">Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.</a> Proceedings of the National Academy of Sciences. 94, 7429-7434 (1997).</li><li>Radicella, J. P., Dherin, C., Desmaze, C., Fox, M. S., Boiteux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+hOGG1,+a+human+homolog+of+the+OGG1+gene+of+Saccharomyces+cerevisiae.">Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae.</a> Proceedings of the National Academy of Sciences. 94, 8010-8015 (1997).</li><li>Banath, J. P., Klokov, D., MacPhail, S. H., Banuelos, C. A., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+gammaH2AX+foci+as+an+indication+of+lethal+DNA+lesions.">Residual gammaH2AX foci as an indication of lethal DNA lesions.</a> BMC Cancer. 10, 4 (2010).</li><li>Banath, J. P., Macphail, S. H., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+sensitivity,+H2AX+phosphorylation,+and+kinetics+of+repair+of+DNA+strand+breaks+in+irradiated+cervical+cancer+cell+lines.">Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.</a> Cancer Res. 64, 7144-7149 (2004).</li><li>Bonner, W. M., 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=[gamma]H2AX+and+cancer.">[gamma]H2AX and cancer.</a> Nat. Rev. Cancer. 8, 957-967 (2008).</li><li>Finney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+Field+Gel+Electrophoresis.">Pulsed Field Gel Electrophoresis.</a> Current protocols in molecular biology. , (2000).</li><li>Electrophoresis, F. I., 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> Pulsed-field gel electrophoresis. , (2000).</li><li>Nowsheen, S., Bonner, J. A., Yang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poly+(ADP-Ribose)+polymerase+inhibitor+ABT-888+reduces+radiation-induced+nuclear+EGFR+and+augments+head+and+neck+tumor+response+to+radiotherapy.">The poly (ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy.</a> Radiotherapy and Oncology. , (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

혜성 분석은 개별 세포를 분석의 고유 한 능력이 있습니다. 이 세포 독성 요원에게 차등 응답을 보여 세포의 subpopulations의 식별에 유리한 것입니다. 몇 실제적인 제한이 고려되어야한다. 개별적으로 평가 될 수있는 세포의 수는 개인에 따라 다를 수 있습니다. 인구 내에서 DNA 손상에 차이가있을 경우 표본의 크기가 증가해야합니다. 괴사 또는 apoptotic 세포의 주된 존재가 에러가 추가되므로 가능한...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 1.5 ML 튜브 </td><td> 산타 크루즈 (Santa Cruz) 생명 공학, Inc의 </td><td> SC-200271 </td><td></td></tr><tr><td> 10X TBE (트리스베이스, 붕소의 산성, EDTA (에틸렌 다이아 민 테트라 초산)) </td><td> 피셔 과학 </td><td> BP13331 </td><td></td></tr><tr><td> 15 ML 튜브 </td><td> 피셔 과학 </td><td> 0553851 </td><td></td></tr><tr><td> 아가로 오스 </td><td> 시그마 </td><td> A5093 </td><td></td></tr><tr><td> 알루미늄 호일 </td><td> 피셔 과학 </td><td> 01213101 </td><td></td></tr><tr><td> 굽​​ 달린 큰 컵 </td><td> 피셔 과학 </td><td> FB102300 </td><td></td></tr><tr><td> 원심 분리기 </td><td> 열 과학 </td><td> 75004261PR </td><td></td></tr><tr><td> 혜성 분석 소프트웨어 </td><td> 혜성 분석 IV 이미지 분석 시스템 </td><td></td><td></td></tr><tr><td> 실린더 </td><td> 피셔 과학 </td><td> 08555F </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> GIBCO </td><td> 15,575 </td><td></td></tr><tr><td> 전기 챔버 </td><td> 열 과학 </td><td> 09528101 </td><td></td></tr><tr><td> 에탄올 </td><td> 피셔 과학 </td><td> A407P4 </td><td></td></tr><tr><td> 형광 현미경 </td><td> Zeiss Axio 비전 </td><td></td><td> 그린 필터 가진 형광 현미경은 충분 </td></tr><tr><td> Hemacytometer </td><td> 피셔 과학 </td><td> 0267152 </td><td></td></tr><tr><td> 낮은 녹는 점 아가로 오스 </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> 마이크로파 </td><td> 시어스 </td><td></td><td></td></tr><tr><td> 인산염은 무료 생리, 칼슘 무료, 마그네슘 버퍼 </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> 전원 공급 장치 </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> 냉장고 </td><td> 시어스 </td><td></td><td></td></tr><tr><td> 지배자 </td><td> 스테이플 </td><td></td><td></td></tr><tr><td> 슬라이드 </td><td> 피셔 과학 </td><td> 12550143 </td><td></td></tr><tr><td> 나트륨 염화물 </td><td> 시그마 </td><td> S7653 </td><td></td></tr><tr><td> 수산화 나트륨 </td><td> 시그마 </td><td> S5881 </td><td></td></tr><tr><td> 나트륨 라 우릴 sarcosinate </td><td> 피셔 과학 </td><td> S529 </td><td></td></tr><tr><td> Sybr 녹색 </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> 쟁반 </td><td> FIS그녀 과학 </td><td> 15242B </td><td></td></tr><tr><td> 트리스베이스 </td><td> 시그마 </td><td> T6066 </td><td></td></tr><tr><td> 트리톤 - X 100 </td><td> 시그마 </td><td> T8787 </td><td></td></tr><tr><td> 트립신 </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> 소용돌이 </td><td> 피셔 과학 </td><td> 02216108 </td><td></td></tr><tr><td> 열탕 목욕 </td><td> 피셔 과학 </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

약물의 수는 DNA 복구 경로를 타겟으로하고 세포가 DNA 손상을 만들어 죽 유도. 따라서, 직접 D​​NA 손상을 측정하기위한 프로세스를 광범위하게 평가되었습니다. 전통적인 방법은 시간이 소요, 비용, 리소스를 집중하고 세포가 복제를 필요로합니다. 반면, 혜성 분석, 단일 세포 겔 전기 영동 분석은 DNA 손상 및 수리를 빠르고 비 침습적, 저렴한, 직접 민감한 측정하기위한 한 방법입니다. DNA 손상뿐만 아니라 DNA 수리의 모든 양식이 강력한 기술을 사용하여 단일 세포 수준에서 시각화 할 수 있습니다. 혜성 분석의 기초 원리는 DNA 손상이 조직을 방해 반면에 그대로 DNA가 높은 주문이다. 손상된 DNA의 아가로 오스 매트릭스에 새어 나온 및 전기 분야의 대상이 때, 부정적인 청구 DNA가 긍정적으로 청구됩니다 음극으로 마이그레이션합니다. 큰 피해 DNA의 가닥 훨씬 핵에서 마이그레이션 할 수 없습니다. DNA 손상더 손상 DNA보다 여행 작은 DNA 조각을 만듭니다. 혜성 분석, 이미지 분석 소프트웨어, 조치하고 핵에서 마이그레이션 DNA와 핵에서 DNA의 전체 형광 강도를 비교합니다. 마이그레이션 DNA의 형광 신호가 DNA 손상에 비례합니다. 긴 밝은 DNA 꼬리 증가 DNA 손상을 의미합니다. 측정 매개 변수 중 일부는 꼬리에있는 DNA의 금액과 꼬리, 꼬리 길이 DNA의 유통 및 DNA의 비율 모두의 척도입니다 꼬리 순간입니다. 이 분석은 DNA 손상의 해상도가 복구가 자리를 차지하게 때문에뿐만 아니라 DNA 복구를 측정 할 수 있습니다. 감도의 제한 diploid 포유류의 세포 1,2 당 약 50 스트랜드 나누기입니다. 같은 etoposide와 같은 DNA 손상 대리인과 치료 세포는 긍정적 인 컨트롤로 사용할 수 있습니다. 따라서 혜성 분석은 DNA 손상을 측정 할 수있는 신속하고 효과적인 절차입니다.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

혜성 분석은 알칼리 불안정한 사이트와 hippocampal 뉴런을 포함한 모든 세포의 DNA에 DNA-DNA/DNA-protein 상호 링크 등 단일 및 이중 가닥 휴식을 검출 효율적인 방법입니다. 방법은 DNA 손상의 양의 차이로 인해 전기 분야에서 DNA의 차동 마이그레이션을 활용합니다.

혜성 분석을 사용하여 Hippocampal 뉴런의 DNA 손상을 시금

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.3K 조회수.  University of Alabama-Birmingham. 혜성 분석은 알칼리 불안정한 사이트와 hippocampal 뉴런을 포함한 모든 세포의 DNA에 DNA-DNA/DNA-protein 상호 링크 등 단일 및 이중 가닥 휴식을 검출 효율적인 방법입니다. 방법은 DNA 손상의 양의 차이로 인해 전기 분야에서 DNA의 차동 마이그레이션을 활용합니다.

비디오: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

유전자 변형을 사용하여 시금 β - 아밀로이드 독성 C. 엘레간스 모델

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

연구

신경과학

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA 손상 생체 외에서 혜성 분석 결과 사용 하 여 평가

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

암 연구학

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip : Microarrayed 인간의 세포에서 DNA 손상을 측정하기위한 높은 처리량 96 글쎄 플랫폼

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

생체공학

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

태아 쥐의 Hippocampal 신경 세포의 분리 및 문화

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

DNA 손상의 평가를 위한 혜성 분석에서 냉동 조직의 사용

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

유전학

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

전신 산화 스트레스의 간접 측정으로 혜성 분석

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

생물학

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

쥐 간에서 DNA 가닥 나누기 및 산화 적 DNA 손상을 측정하기위한 생체 알카라인 혜성 분석 및 효소 수정 알카라인 혜성 분석에서

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

세포 DNA 손상 평가를 위한 고처리량 혜성 분석 접근법

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app.jove.com/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

세포의 DNA 손상 감지를 위한 Comet Assay

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...