Name: evaluating in vitro dna damage using comet assay
Uploaded: 2017-10-11T14:00:00.000+00:00
Duration: 10 min 43 s
Description: Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

이 연구는 NIH, NCI, CCR의 교내 연구 프로그램에 의해 지원 되었다. 모든 저자는 NIH, NCI, 및 CCR에서 교내 연구 보조금을 받았다.

1. 시 약 준비 <ol><li>1 x PBS <ol><li>900 mL dH 2 O 100 mL 10 x PBS를 희석 하 고 pH 미터를 사용 하 여 7.4에 pH를 조정. 실내 온도에 상점.</li>
	</ol></li> <li>세포 솔루션 (LS) <ol><li>2.5 M NaCl, 100 m m disodium EDTA, 10 mM Tris 자료, 및 900 mL dH 2 O 200 m m NaOH 준비, 그것은 일반적으로 완전 해산을 혼합 수 있도록 약 20 분 걸립니다. 10 pH 미터를 사용 하 여 pH를 조정 합니다. 추가 1% 나트륨 lauryl sarcosinate 및 1% 트라이 톤 X-100 1000 mL를 최종 볼륨을 조정. 사용 하기 전에 적어도 30 분 동안 4 ° C에 쿨.</li>
	</ol></li> <li>알칼리 성 전기 이동 법 솔루션 (AES), pH &#62; 13 <ol><li>200 m m NaOH와 1 m m disodium EDTA 800 mL dH 2 O. 조정 산도 준비 하 고 pH는 &#62; 13. 1000 mL를 최종 볼륨을 조정 합니다. 사용 하기 전에 신선한 확인 하 고 사용 하기 전에 적어도 30 분 동안 4 ° C에 냉각.</li>
	</ol></li> <li>중립 전기 sollution (NES) <ol><li>빙 초 산 100 mM Tris 기지와 300 mM 아세트산 나트륨 1000 mL dH 2 O. 조절 pH 9.0 혼합 하 여 준비 1000 mL 중립 전기 이동 법 버퍼. 사용 하기 전에 적어도 30 분 동안 4 ° C에 쿨.</li>
	</ol></li> <li>DNA 강수량 솔루션 (DPS) <ol><li>10 mL 7.5 M 암모늄 아세테이트 주식의 준비. DNA 강수량 솔루션 50 ml, 6.7 mL 7.5 M 염화 아세테이트 43.3 mL 95% 에탄올 혼합. 실내 온도에 상점.</li>
	</ol></li> <li>얼룩 솔루션 <ol><li>추가 1 µ L 10000 녹색 형광 핵 산 얼룩 (예를 들어, SYBR 녹색) 30 mL 트리 스-EDTA 버퍼 (10 mM Tris HCl, 1 m m disodium EDTA, pH 7.4)에 게 x 4 ° C. 보호에서 빛.</li>
	</ol></li> <li>1% 낮은 녹는 agarose <ol><li>1% 낮은 녹는 포인트 agarose (1 g 100 mL dH 2 O) 전자 레인지에 녹여. 모든 15-20 s는 agarose는 완전히 녹은 것을 확인 하는 agarose를 소용돌이 친다. 사용 하기 전에 적어도 20 분 동안 37 ° C 물 목욕에는 agarose를 놓습니다.</li>
	</ol></li> <li>미리 따뜻한 피 펫 팁 <ol><li>P200 피 펫 팁의 좁은 끝 3 mm와 37 ° C에서 따뜻한 agarose를 pipetting 전에 잘라.</li>
	</ol></li></ol> 2. 혜성 슬라이드 준비 <ol><li>코팅 슬라이드 <ol><li>는 agarose는 완전히 녹은 때까지 또는 2-3 분 동안 전자 레인지에 녹여 1 %agarose (1 g 100 mL dH 2 O). 유리 현미경 슬라이드는 agarose 찍어와 보풀 지우기를 사용 하 여 슬라이드의 한 면을 닦아 내십시오.</li>
		<li>누워 건조 또는 빠른 건조를 위해 50 ° C에가 열 하는 평평한 표면에 슬라이드; 건조 후 투명 agarose 필름을 형성 한다. 사용 하기 전에 37 ° C에 코팅된 슬라이드 배치.</li>
	</ol></li> <li>단일 셀 정지의 준비 <ol><li>문화와 치료 glioma 셀 <ol><li>DMEM 햄 F-12 매체 10 보충에 U251 MG 셀 문화 %FBS, 100 U/mL 페니실린, 및 37 & #에서 10 µ g/mL 스 176; 5% CO 2와 C.</li>
			<li>3 분, 1 mL의 트립 신을 사용 하 여 셀을 소화 하 고 중화 FBS와 DMEM 햄 F-12 매체를 사용 하 여 트립 신. 수집 15 mL 튜브에 4 분에 대 한 300 x g에서 회전, 매체, 발음 및 1 x PBS에서 2 x 10 5 셀/mL에서 셀 중단. 
			참고: 셀 샘플 분석 결과 시작 하기 전에 바로 준비 해야 하 고 모든 샘플에서에서 DNA 손상을 방지 하기 위해 어두운 또는 흐리게 환경에서 처리 되어야 합니다.</li>
			<li>결합 세포 현 탁 액 비율 (37 ° C)에서 1% 녹은 낮은 융 점 agarose 1:10 (v/v), 아래로, pipetting으로 부드럽게 혼합 하 고 즉시 슬라이드에 30 µ L 플라스틱. 사용의 측면에 얇은 층의 형성을 위해 agarose/셀 혼합 확산 피 펫 팁.</li>
			<li>10 분 30 분 하 고 시간을 증가 향상 높은 습도 환경에서 샘플의 준수 어둠 속에서 4 ° C에서 플랫 슬라이드 장소.</li>
			<li>담가 하룻밤 1 h에 대 한 어둠 속에서 4 ° c LS 슬라이드.</li>
		</ol></li></ol></li></ol> 3. 단일 셀 전기 <ol><li>알칼리 (3.2 단계)를 진행 또는 중립 (단계 3.3) 혜성 시험</li> <li>알칼리 혜성 분석 결과 대 한 <ol><li>부드럽게 LS에서 슬라이드를 제거, 배수 초과 버퍼, 그리고 부드럽게 4에서 1 h 동안 AES에 젖어 ° C DNA 해제 수 있도록입니다. 어둠 속에서 슬라이드를 계속.</li>
		<li>전기 슬라이드 트레이에 추가 미리 냉장된 AES (이 전기 단위의 크기에 따라 달라 집니다) 슬라이드 위에 0.5 c m를 초과 하지 않는, 내부 슬라이드와 모자와 함께 커버. 전원 공급 전압 1 V/cm (전극 사이의 길이)을 설정 하 고 4 시 30 분에 대 한 실행 ° c.</li>
		<li>슬라이드에서 드레인 과잉 전기 솔루션입니다. DH 2 O 각 실 온에서 5 분에 두 번 슬라이드를 담가 부드럽게.</li>
		<li>부드럽게 슬라이드 실 온에서 5 분 동안 70% 에탄올에 젖어. 4 단계를 수행 하십시오.</li>
	</ol></li> <li>중립 혜성 분석 결과 대 한 <ol><li>부드럽게 LS에서 슬라이드를 제거 초과 버퍼, 배수와 부드럽게 4 시 30 분에 대 한 NES에 젖어 ° C. 계속 어둠 속에서 슬라이드.</li>
		전기 이동 법 슬라이드 트레이에서 <li>추가 미리 냉장된 중립 전기 이동 법 버퍼 (이 전기 단위의 크기에 따라 달라 집니다) 슬라이드 위에 0.5 c m를 초과 하지 않는, 내부 슬라이드와 모자와 함께 커버. 전원 공급 전압 1 V/cm (전극 사이의 길이)을 설정 하 고 4에서 45 분 동안 실행 ° c.</li>
		<li>드레인 슬라이드에서 초과 버퍼입니다. 실 온에서 30 분에 대 한 DPS에서 슬라이드를 담가 부드럽게.</li>
		<li>부드럽게 실 온에서 30 분 동안 70% 에탄올에 슬라이드를 담가. 4 단계를 수행 하십시오.</li>
	</ol></li></ol> 4. 혜성 슬라이드 얼룩 <ol><li>어둠 속에서 10-15 분 동안 37 ° C에서 슬라이드 건조.</li>
	<li>장소 50-100 µ L 녹색 형광 핵 산 각에 솔루션 얼룩 말린 agarose 및 어둠 속에서 실 온에서 15 분 동안 얼룩.</li>
	<li>DH 2 O에서에서 간단히 슬라이드를 헹 구 고 완전히 어둠 속에서 37 ° C에서 건조. 이미지 수집 및 분석 진행.</li>
</ol> 5. 이미지 수집 및 분석 참고: 시각화와 DNA 나누기의 정량화는 기반 epifluorescence 현미경 검사 법 및 혜성 분석 소프트웨어 (재료의 표 참조) 12 .
<ol><li>슬라이드 홀더를 가진 현미경에 슬라이드를 놓습니다. Agarose 젤 대물 렌즈에 직면 하 고 있는지 확인 합니다. 무작위로 10 배 대물 렌즈와 형광 현미경을 사용 하 여 스테인드 혜성 슬라이드에서 이미지를 캡처하십시오. 가장자리 및 어떤 공기 방울 주변 지역.</li>
	<li>확인 각 혜성 꼬리 수평으로 배포 됩니다. 혜성 머리 왼쪽과 오른쪽에서 꼬리에서 발생 합니다.</li>
	<li>어두운 배경에 밝은 DNA 얼룩과 이진 TIF 형식에 각 그림을 저장합니다. 소프트웨어를 사용 하 여 이미지 로드는 &#34; 파일 분석을 선택 &#34; 도구 모음의 왼쪽에 있는 버튼. 이미지 보기 창 ( 그림 1)를 표시 합니다.</li>
	<li>측정 프레임 화면에 그릴 하 고 셀의 혜성에 그것의 크기를 조정 합니다. 클릭에 &#34; 조정 &#34; 머리, 혜성, 그리고 이미지를 따라 꼬리의 임계값 설정 버튼을 클릭에 &#34; 측정을 시작 &#34; 단추 ( 그림 1).</li>
	<li>프레임을 사용 하 여 셀을 선택 하 고 마우스로 클릭 하 여 측정을 활성화는 &#34; 시험의 혜성 &#34; 버튼, 강도 이미지에 나타난다는 &#34; 프로필 &#34; 선택한 측정 매개 변수 창. 클릭 하 여 결과 저장할 수 있는 &#34; 결과 저장 &#34; 단추 ( 그림 1). 
	참고: 소프트웨어 혜성 꼬리, 꼬리 DNA, 꼬리 순간 (TM)와 올리브 꼬리 순간 (OTM)의 비율의 길이 포함 하 여 매개 변수를 계산 합니다. 꼬리 순간 다음과 같이 수식으로 계산 된다: <img alt="Equation 1" src="/files/ftp_upload/56450/56450eq1.jpg" /> <img alt="Equation 2" src="/files/ftp_upload/56450/56450eq2.jpg" /></li> <li>50 셀 분석 치료 당.</li>
</ol>

<ol>
	<li>Ostling, O., Johanson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochem Biophys Res Commun. 123 (1), 291-298 (1984).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells.">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp Cell Res. 175 (1), 184-191 (1988).</li><li>Tice, R. R., 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=Single+cell+gel/comet+assay:+guidelines+for+in+vitro+and+in+vivo+genetic+toxicology+testing.">Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.</a> Environ Mol Mutagen. 35 (3), 206-221 (2000).</li><li>Shah, A. J., Lakkad, B. C., Rao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+in+lead+treated+human+lymphocytes+evaluated+by+micronucleus+and+comet+assays.">Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.</a> Indian J Exp Biol. 54 (8), 502-508 (2016).</li><li>Azqueta, A., 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+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch Toxicol. 87 (6), 949-968 (2013).</li><li>Goldstein, M., Kastan, 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=The+DNA+damage+response:+implications+for+tumor+responses+to+radiation+and+chemotherapy.">The DNA damage response: implications for tumor responses to radiation and chemotherapy.</a> Annu Rev Med. 66, 129-143 (2015).</li><li>Gavande, N. 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=DNA+repair+targeted+therapy:+The+past+or+future+of+cancer+treatment?.">DNA repair targeted therapy: The past or future of cancer treatment?.</a> Pharmacol Ther. 160, 65-83 (2016).</li><li>Torgovnick, A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+mechanisms+in+cancer+development+and+therapy.">DNA repair mechanisms in cancer development and therapy.</a> Front Genet. 6, 157 (2015).</li><li>Weston, V. J., 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+PARP+inhibitor+olaparib+induces+significant+killing+of+ATM-deficient+lymphoid+tumor+cells+in+vitro+and+in+vivo.">The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.</a> Blood. 116 (22), 4578-4587 (2010).</li><li>Brown, J. S., O'Carrigan, B., Jackson, S. P., Yap, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+Repair+in+Cancer:+Beyond+PARP+Inhibitors.">Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.</a> Cancer Discov. 7 (1), 20-37 (2017).</li><li>Lu, Y., 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=Chemosensitivity+of+IDH1-Mutated+Gliomas+Due+to+an+Impairment+in+PARP1-Mediated+DNA+Repair.">Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.</a> Cancer Res. 77 (7), 1709-1718 (2017).</li><li>Konca, K., 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+cross-platform+public+domain+PC+image-analysis+program+for+the+comet+assay.">A cross-platform public domain PC image-analysis program for the comet assay.</a> Mutat Res. 534 (1-2), 15-20 (2003).</li><li>Valverde, M., Rojas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+occupational+biomonitoring+using+the+Comet+assay.">Environmental and occupational biomonitoring using the Comet assay.</a> Mutat Res. 681 (1), 93-109 (2009).</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:+principles,+applications,+and+limitations.">The comet assay for DNA damage and repair: principles, applications, and limitations.</a> Mol Biotechnol. 26 (3), 249-261 (2004).</li><li>Karbaschi, M., Cooke, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+for+the+high-throughput+processing+of+slides+for+the+comet+assay.">Novel method for the high-throughput processing of slides for the comet assay.</a> Sci Rep. 4, 7200 (2014).</li></ol>

저자는 공개 없다.

혜성 분석 결과 세포 수준에서 단일 및 이중 가닥 DNA 나누기를 측정 하는 효율적인 도구 이다. 분석 결과 연구에 관한 genotoxicity 및 biomonitoring13, 기본 병 변, DNA crosslinks, 약물 개발 및 알칼리 민감한 사이트에서까지 "황금 표준"으로 널리 적용 되었습니다. 현재 연구에서 우리가 알칼리와 중립 혜성 분석 실험에 대 한 두 가지 단계별 프로토콜 각각 보였다. 결합 하 여 단일 셀 전기 ...

혜성 분석 결과 처음 개발 되었다 Ostling와 요한슨에 의해 1984 년에 DNA의 마이그레이션 중립 조건1아래 핵에서 조각으로. 기술은 나중 싱 외., 보여주는 알칼리 상태를 실질적으로 특이성 및 재현성 분석 결과2의 증가 의해 개발 되었다. 그 이후, 중립 혜성 분석 결과 주로 데 반면에 알칼리 성 혜성 분석 결과 DNA 손상, 싱글 등의 작은 금액에 대 한 더 민감한 이중 가닥 DNA 틈, 알칼리 정한 사이트, DNA-DNA 이중 가닥 DNA 틈, 검색 또는 DNA-단백질 cross-linking 그리고 DNA 단일 가닥 나누기 불완전 절단과 관련 된 사이트3,4복구. 두 분석 된 DNA의 시각화를 허용 하 고 양적 DNA 손상을 평가 하는 간단한 방법을 제공. 혜성 분석 결과 생체 외에서 그리고 vivo에서 유전 독성 연구를 위한 중요 한 방법으로 간주 되 고 초기 약물 후보 선택, 환경 모니터링, 인간의 biomonitoring 다른 연구 분야에 적용 됩니다. 그리고 기본 DNA 손상에 연구 하 고5를 복구.
분석 결과의 원리가 이다 전기 분야에서 조각난된 DNA는 핵양체 몸에서 (일컬어 "혜성 머리") 마이그레이션합니다와 agarose 젤 (일컬어 "혜성 꼬리")에서 DNA 얼룩을 형성. 뉴클레오티드 얼룩과 DNA 손상의 범위 "혜성"이 단일 셀 전기 이동 법에 의해 형성을 분석 하 여 측정할 수 있습니다. 꼬리 순간의 계산 더 비교 다른 실험적인 그룹 간의 DNA 손상 시킬 수 있습니다. DNA 손상 탐지의 전통적인 방법에 비해, 혜성 분석 결과 이며 직접, 민감한, 저렴 한, 상대적으로 간단한.
방사선 치료와 chemotherapies 단일 가닥을 생성 하 여 암 치료에 대 한 일반적인 전략은와 이중 가닥 DNA는 염색체6에 나누기. DNA 복구 억제제의 최근 발전 조합 화학요법에 의해 더 효과적인 차적인 효과 허용 하 고 따라서 잠재적으로 빈 혈, 감염, 골 수 억제7, 같은 조직의 부작용을 감소 8.이 연구에서 우리는 많은 (ADP-ribose) (PARP) 억제제, olaparib (Ola)9의 조사 했다. PARP 풍부한 핵 단백질 이며 많은 (ADP-ribose) 폴리머10을 형성 하 여 DNA 기본 절단 복구에 대 한 책임입니다. Temozolomide (TMZ)는 구두로 사용 가능한 alkylating 에이전트 이며 glioma 환자 치료를 위해 널리 이용 되는. DNA 손상 척도를 혜성 분석 결과 사용 하 여 우리는 olaparib/temozolomide 조합 치료 glioma, 치료 하는 효과적인 전략을 제안 glioma 세포에서 DNA 손상 향상 결합 olaparib temozolomide 깊이 설명 temozolomide 혼자11와 비교.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x PBS(Ca&lt;sup&gt;++&lt;/sup&gt;, Mg&lt;sup&gt;++&lt;/sup&gt; free)</td><td>TEKnova</td><td>P0196</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>EDTA</td><td>TEKnova</td><td>E0308</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>Sodium lauryl sarcosinate</td><td>Sigma</td><td>L7414</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>32318</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>695092</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma</td><td>A1542</td><td></td></tr><tr><td>SYBR Green</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Low melting point agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500</td><td></td></tr><tr><td>95% ethanol</td><td>WARNER-GRAHAM</td><td>#64-17-5</td><td></td></tr><tr><td>Trypsin</td><td>GIBICO</td><td>25300-054</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glass tissue slides</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>63422-11</td><td></td></tr><tr><td>Kimwipes</td><td>KIMberly-Clark</td><td></td><td></td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>DENVILLE</td><td></td><td></td></tr><tr><td>Pipette Tips</td><td>SHARP</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microwave</td><td>Avanti</td><td></td><td></td></tr><tr><td>Waterbath</td><td>PRECISION</td><td></td><td></td></tr><tr><td>Horizontal electrophoresis chamber</td><td>TREVIGEN</td><td>Cometassay ES II</td><td></td></tr><tr><td>Power supply</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Incubator</td><td>Quincy Lab</td><td>Model 12-140E</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Micropipettor</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

evaluating in vitro dna damage using comet assay

DNA 손상의 수명 동안은 각 셀에 대 한 일반적인 현상이 며 genomic DNA의 화학 구조에의 한 변경으로 정의 됩니다. 암 치료, 라디오-및 화학 요법 같은 엄청난 양의 추가 DNA 손상, 세포 주기 검거와 apoptosis 제한 암 진행을 이끌어 소개. 실험적인 암 치료 기간 동안 DNA 손상의 정량적 평가 차적인 에이전트의 효율성을 정당화 하기 위해 중요 한 단계입니다. 이 연구에서 우리는 단일 셀 전기 이동 법 분석 결과, 일컬어 혜성 분석 결과, 단일 계량 수 있는에 집중 하 고 이중 가닥 DNA 생체 외에서휴식. 혜성 분석 결과 DNA 손상 부 량 효율적이 고, 실행 하기 쉬운 낮은 시간/예산 요구 및 높은 재현성. 여기, 우리 U251 glioma 셀에 olaparib/temozolomide 조합 치료의 차적인 효과 평가 하 여 전 임상 연구에 대 한 혜성 분석 결과의 유틸리티를 강조 표시 합니다.

현재 프로토콜 혜성 분석 결과 실행 및 데이터 분석 (그림 1)에 대 한 단계별 워크플로 설명합니다. 알칼리와 중립 혜성 분석 실험 결과 나타났다 U251 세포 독 소 루비 취급 (1 µ M, 20 h)의 혜성 꼬리 더 오래 되었고 더 높은 DNA 강도, 화학 요법 (그림 2)으로 조각난된 DNA의 상당한 축적을 제안 했다.
<p class="jove_content" fo:keep-together....

Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Evaluating In Vitro DNA Damage Using Comet Assay

DNA 손상 생체 외에서 혜성 분석 결과 사용 하 여 평가

혜성 분석 결과 단일과 이중 가닥 DNA를 포함 하 여 DNA 손상 나누기를 검색 하는 효율적인 방법입니다. 알칼리와 중립 혜성 분석 실험 화학 요법의 치료 효과 평가 하기 위해 암 세포의 DNA 손상을 측정을 설명 합니다.

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 ...

evaluating-in-vitro-dna-damage-using-comet-assay

Research

JoVE Journal

Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay

37.1K 조회수.  National Cancer Institute. 혜성 분석 결과 단일과 이중 가닥 DNA를 포함 하 여 DNA 손상 나누기를 검색 하는 효율적인 방법입니다. 알칼리와 중립 혜성 분석 실험 화학 요법의 치료 효과 평가 하기 위해 암 세포의 DNA 손상을 측정을 설명 합니다.

비디오: Evaluating In Vitro DNA Damage Using Comet Assay

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

위쪽으로 이동: 간단 하 고 유연한 생체 외에서 3 차원 침공 분석 실험 프로토콜

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

연구

암 연구학

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

분석 결과 돋 아 회전 타원 체를 사용 하 여 상 상속 Hemangioblastoma Neovascularization의 생체 외에서 성능을 평가 하는 포괄적인 절차

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

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

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

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

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 extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

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 ...

신경과학

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 ...

Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy

Source: Lu, Y., et al. <a href="https://www.jove.com/v/56450">Evaluating In Vitro DNA Damage Using Comet Assay</a> J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.

알칼리성 단세포 겔 전기영동: 화학요법 후 개별 암세포의 DNA 손상을 정량적으로 추정하기 위한 민감한 기술

Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

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 ...