Name: immunofluorescence microscopy of γh2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks
Uploaded: 2017-11-03T14:00:00.000+00:00
Duration: 10 min 47 s
Description: Watch this Scientific Journal Video about Immunofluorescence Microscopy of Î³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

프로젝트는 독일 호세 카레라스 백혈병 재단 (DJCLS 14 R/2017)에 의해 지원 되었다.

여기에서 설명한 모든 방법을 의료 학부 임 하이델베르크 대학 윤리 위원회 II에 의해 승인 되었습니다. 모든 개인에서 동의 얻어 작성.
 1. 재료의 준비 <ol><li>응고 제 재고 솔루션: 0.9% 염화 나트륨에에서 mL 당 200 I.U. 덤플링의 항 응 혈 약 재고 솔루션을 준비. 컬렉션 튜브의 각 채우기 (볼륨 그릴 9 mL) 혈액 또는 골 수 샘플의 철수 하기 전에 항 응 혈 약 재고 솔루션의 2 mL와.</li>
	<li>적혈구 세포 솔루션: 레드 셀 염화 암모늄 82.91 g, 7.91 g 염화 중 탄산염, 그리고 pH 8.0에서 에이전트를 용 해 하 여 0.5 M ethylenediaminetetraacetic 산 성 해결책의 2 mL에 lysis 솔루션 x 10 준비 1의 전체 볼륨에 대 한 이중 증 류 물 L.</li>
	<li>고정 솔루션: 추가 8.3 µ L 360 mg paraformaldehyde (PFA) 결정 튜브에서에 1 M 수산화 칼륨 해결책의. 튜브 및 열 36 %PFA 솔루션의 1 mL를 95 ° C에가 열 블록에 튜브의 1 mL 마크를 버퍼링 하는 인산 염 분 (PBS)로 튜브를 채우십시오. 15 mL의 용량을 가진 튜브를 1 mL 36 %PFA 솔루션을 전송 합니다. 8 mL PBS 1 mL 36 %PFA 솔루션 4 %PFA 재고 솔루션의 9 mL를 추가 합니다. 셀의 고정을 위한 4 %PFA 재고 솔루션 및 저장소까지-18 ° C에서 사용 하는 약 수. 
	주의: 1) 수산화 칼륨 해결책은 부식성 이다. 눈과 피부 보호를 알고 있어야 합니다. 2) PFA 발암 성 이다입니다. 피부 접촉 및 흡입 하지 마십시오. 준비는 후드 아래 고정 솔루션.</li>
	<li>Permeabilization 솔루션: 추가 50 µ L Octoxinol 9 ~ 50 mL PBS 약 0.1%의 솔루션을 Octoxinol 9.</li>
	<li>차단 솔루션: 5%와 2 %6에서 PBS와 저장소 단백질 블로킹 제를 혼합 하 여 차단 하는 솔루션 준비 &#8211; 8 ° c.</li>
</ol> 2. 샘플의 준비 <ol><li>섬유 아 세포 세포 배양에서: 셀 라인 NHDF 습도 5%에서의 인간의 섬유 아 세포를 배양 10% 태아 종 아리 혈 청, 4mm 보충 RPMI 1640 매체에서 37 ° C에서 CO 2 분위기 글루타민, 그리고 1% 페니실린/스입니다.
	<ol><li>준비는 현미경 슬라이드의 점착 섬유 아 세포 <ol><li>플라스 크 (75 cm 2 성장 지역) 기 하 급수적으로 성장 하는 NHDF 섬유 아 세포에서에서 매체를 제거 합니다. 플라스 크에 10 mL PBS를 추가 합니다. 수동으로 1 분에 대 한 플라스 크를 흔들어 부착 섬유 아 세포를 부드럽게 씻어</li>
			<li>는 PBS를 제거 하 고 플라스 크에 트립 신 1 mL를 추가 합니다. 부드럽게 모든 부착 셀 트립 신에 의해 보호 됩니다 있도록 플라스 크를 흔들어. 플라스 크에서는 트립 신을 제거 합니다. 37 5 분 동안 인큐베이터에 플라스 크를 넣어 ° c.</li>
			<li>는 인큐베이터에서 플라스 크 고 플라스 크에 10 mL RPMI 1640 매체를 추가. 매체는 섬유 아 세포를 분산 여러 번 위아래 플라스틱.</li>
			<li>각 현미경 슬라이드의 두 필드에 분산 된 섬유 아 세포의 0.3 mL를 플라스틱 하 고 슬라이드 하는 섬유 아 세포 준수 24 h에 대 한 인큐베이터에 슬라이드를 넣어. ΓH2AX 및 섬유 아 세포의 핵에서 53BP1의 immunostaining에 대 한 이동 단계 3.1.</li>
		</ol></li></ol></li> <li>환자 샘플 <ol><li>혈액 샘플: 항 응 혈 약 재고 솔루션의 2 mL와 함께 가득 차 있었다 컬렉션 튜브를 사용 하 고 튜브로 7 mL 혈액을 추가. 하룻밤 실 온 (즉, 18-20 ° C)에서 샘플을 저장 합니다. 다음 날, 분리 밀도 그라데이션 원심 (2.2.3 단계)에 의해 단 세포 휴식 G0/G1 단계.</li>
		<li>골 수 샘플: 항 응 혈 약 재고 솔루션 (단계 1.1)의 2 mL와 함께 가득 차 있었다 컬렉션 튜브를 사용 하 고 튜브로 7 mL 골 추가. 하룻밤 실 온 (즉, 18-20 ° C)에서 샘플을 저장 합니다. 다음 날, 단 세포 밀도 그라데이션 원심 (2.2.3 단계)에 의해 휴식 G0/G1 단계 분리. CD34 microbeads를 사용 하 고 셀 CD34 + 세포의 고립에 대 한 분리 열.</li>
		<li>조밀도 기온 변화도 원심 분리 하 여 단 세포의 고립 
		참고: 단 세포 밀도 그라데이션 원심 17 , , 18 19에 의해 혈액과 골 수 샘플에서 격리 됩니다.
		<li>Dilute PBS와 1: 1의 비율로 heparinized 혈액 샘플 및 PBS와 1: 3의 비율로 heparinized 골 수 샘플 <ol>. 그려서 그들 피펫으로 밖 두 번 셀을 부드럽게 중단.</li>
			<li>신선한 튜브를 밀도 그라데이션 매체의 볼륨을 추가합니다. 부드럽게 희석된 혈액 또는 골 수 밀도 그라데이션 매체 위에 레이어. 두 레이어 혼합에 아닙니다 돌.</li>
			<li>원심 관 실 온에서 30 분 동안 하 고 400 x g. 피펫으로와 위 플라즈마 레이어를 그립니다. 다음 신중 하 게 밀도 그라데이션 매체 위에 레이어 단 셀 수확. 단 세포 신선한 튜브로 전송.</li>
			<li>튜브에 단 세포를 PBS의 적어도 3 개의 볼륨을 추가합니다. 그려서 그들 피펫으로 밖 두 번 부드럽게 세포를 일시 중단 합니다. 실내 온도 400 x g.에서 10 분 동안 튜브 원심</li>
			<li>스핀 후는 상쾌한을 제거 하 고 4 ° C, 적혈구 세포 솔루션 x 1의 10 mL을 추가. 그려서 그들 피펫으로 밖 두 번 셀을 부드럽게 resuspend 고 5 분 동안 얼음에 튜브를 놓습니다. 다음 4 ° C에서 30 mL PBS를 추가 하 고 실내 온도 400 x g.에서 10 분 동안 튜브 원심</li>
		</ol></li> <li>준비는 cytospins의 <ol><li>(즉, γH2AX 면역 형광 염색 법에 대 한 하나의 cytospin 및 γH2AX 및 53BP1에 대 한 하나의 cytospin 환자 샘플의 단 세포와 두 cytospins 준비 면역 형광 염색 결합) 원심 분리 (300 x g, 10 분) ( 그림 3 및 4) 각 준비에 대 한 10 5 셀 x 1.0의 의해.</li>
		</ol></li></ol></li></ol> 3. γH2AX 및 53BP1 면역 형광 염색 <ol><li>고정 및 permeabilization: 4%의 200 µ L 셀 해결 PFA 10 분. 고정, 후 30 mL PBS의 각 실험실 통에 5 분을 세 번 셀을 부드럽게 씻어. 다음 세포를 permeabilize 0.1%의 200 µ L을 10 분에 대 한 Octoxinol 9 씻어 셀 부드럽게 각 실험실 통에 5 분 30 mL 5% 차단 솔루션의와 세 번. 1 헤에 대 한 솔루션을 차단 하는 신선한 5%의 30 mL에서 세포를 차단</li>
	<li>항 체를 가진 외피 <ol><li>품 어 마우스 안티 γH2AX 단일 클로 널 항 체 (1: 500)에 포함 된 셀의 한 준비와 마우스 안티 γH2AX 단일 클로 널 항 체 (1: 500)에 포함 된 셀의 다른 준비를 하 고 4에서 하룻밤 polyclonal 토끼 안티-53BP1 항 체 (1: 500) ° c.</li>
		<li>인큐베이션 세척 세포 부드럽게 실험실 셰이 커에 각 5 분 2% 차단 솔루션의 30 mL로 3 시간 후.</li>
		<li>차단 솔루션을 제거 하 고 Alexa488 활용 된 염소 반대로 마우스 2 차 항 체 (1: 500)에 포함 된 셀의 첫 번째 준비와 Alexa488 활용 된 염소 반대로 마우스 이차 항 체 (셀의 두 번째 준비 품 어 1: 500)와 Alexa555 활용 된 당나귀 안티 토끼 2 차 항 체 (1: 500) 실 온에서 1 h.</li>
	</ol></li> <li>장착 매체: 셀으로 슬라이드를 그릇에 넣고 셀 부드럽게 실험실 셰이 커에 각 5 분 30 mL의 PBS로 3 회 세척. PBS를 제거 하 고 장착 매체 4, 6-diamidino-2-phenylindole를 포함 하는 셀을 탑재. 조심 스럽게 넣어 장착 매체 위에 coverslip 없음 기포는 포함 된. 형광 현미경으로 세포를 분석 하기 전에 장착 매체의 강화에 대 한 적어도 3 h 기다립니다.</li>
</ol> 4. ΓH2AX 및 53BP1의 분석 Foci <ol><li>형광 현미경 검사 법: 100 X 목표에 이미징 동안 DAPI, 알 렉 사 488, Cy3 필터를 갖춘 형광 현미경으로 세포 핵에서 γH2AX 및 53BP1 foci 분석 확대입니다. 카메라와 함께 이미지를 기록 하 고 적절 한 이미징 소프트웨어로 이미지 처리.</li>
</ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+DNA+damage+after+reduced+and+conventional+absorbed+radiation+doses+using+3rd+generation+dual-source+CT+technology.">Leukocyte DNA damage after reduced and conventional absorbed radiation doses using 3rd generation dual-source CT technology.</a> Eur J Radiol Open. 3, 134-137 (2016).</li><li>Rothkamm, K., Balroop, S., Shekhdar, J., Fernie, P., Goh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+DNA+damage+after+multi-detector+row+CT:+a+quantitative+biomarker+of+low-level+radiation+exposure.">Leukocyte DNA damage after multi-detector row CT: a quantitative biomarker of low-level radiation exposure.</a> Radiology. 242 (1), 244-251 (2007).</li><li>Lobrich, 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=In+vivo+formation+and+repair+of+DNA+double-strand+breaks+after+computed+tomography+examinations.">In vivo formation and repair of DNA double-strand breaks after computed tomography examinations.</a> Proc Natl Acad Sci U S A. 102 (25), 8984-8989 (2005).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fixatives+for+immunostaining.">Cell fixatives for immunostaining.</a> Methods Mol Biol. 588, 55-61 (2010).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+cell+membranes.">Permeabilization of cell membranes.</a> Methods Mol Biol. 588, 63-66 (2010).</li><li>Rothkamm, K., Lobrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+lack+of+DNA+double-strand+break+repair+in+human+cells+exposed+to+very+low+x-ray+doses.">Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.</a> Proc Natl Acad Sci U S A. 100 (9), 5057-5062 (2003).</li></ol>

저자는 공개 없다.

면역 형광 검사 현미경 검사 법의 γH2AX와 53BP1 형성 및 연구 분야의 넓은 스펙트럼에서 DSB의 수리 분석을 위한 유용한 방법입니다. 실험의 결과 좌우 하는 중요 한 매개 변수는 세포 주기, 고정 및 permeabilization 세포, 항 체, 그리고 하드웨어의 선택과 형광 현미경의 소프트웨어 사용 하는 에이전트의 단계.
ΓH2AX foci 패턴에 세포 주기의 영향 정상 섬유 아 세포 세포 라인 NHDF의 기...

DNA는 지속적으로 내 생에 의해 손상 (예를 들어, 복제 스트레스, 반응성 산소 종, DNA의 본질적인 불안정성) 및 외 인 (예, 화학 기, 방사선) 소스 (그림 1)1,2 ,,34. DNA 손상, 중 DNA 두 배 물가 틈 (DSB) 특히 심각한 병 변 그리고 세포 죽음 또는 발암을 일으킬 수 있습니다. 약 50 DSB는 세포와 세포 주기5당 발생할 수 있습니다. 포유류 세포, 동종 재결합 (HR) 및 nonhomologous 끝-결합 (NHEJ) 개발 DSB (그림 2)의 복구에 대 한 주요 경로. 인사 늦게 S/g 2 단계에서 발생 하 고 그대로 동생을 사용 하 여 잠재적으로 오류 없이 복구에 대 한 템플릿으로 chromatid. 비교에서는, NHEJ 기본적인 쌍을 추가 또는 깨진된 끝의 결 찰 하기 전에 절제 수 있습니다 및 잠재적으로 돌연변이 세포 주기입니다. 또한, 대체 최종 합류 NHEJ 결핍6,7시 느린 돌연변이 백업 복구 메커니즘으로 약혼 수 있습니다.
DSB 각 DSB 주위 여러 megabase 쌍의 영역에 떠들고 139에서 히스톤 H2AX의 인 산화를 유도. 형성 핵 foci γH2AX foci 이름이 되며 면역 형광 검사 현미경 검사 법8감지. ΓH2AX 추가 DSB 반응 단백질의 신규 모집을 촉진 하 고 개장 하는 chromatin, DNA 수리, 및 신호 변환9에 관여. 각 γH2AX 초점을 나타내는 단일 DSB 간주 됩니다, 면역 형광 검사 현미경 검사 법에 의해 DSB의 정량화는 가능,이 암 세포 선 및 환자 표본10,11, 에서 입증 되었습니다. 12 , 13 , 14. 53BP1 (p53 바인딩 단백질 1) DSB 수리 중재에 또 다른 주요 단백질 이다. 그것은 DSB 반응 단백질, 검사점 신호, 그리고 DSB 끝15synapsis의 채용에 관여. 또한, 53BP1 DSB 복구 경로 선택에서 중요 한 역할을 하고있다. 그것은 시간 동안 방해16NHEJ 향해 DSB의 수리를 트리거합니다. ΓH2AX 및 DSB 수리에 53BP1의 진정한 기능을 고려 하면 γH2AX 및 53BP1 면역 형광 검사 현미경 검사 법에 의해의 동시 분석 형성 및 DSB의 복구의 정확한 분석을 위한 유용한 방법 있을 수 있습니다.
이 원고는 세포 핵에 γH2AX와 53BP1의 면역 형광 검사 현미경 검사 법을 수행 하기 위한 단계별 프로토콜을 제공 합니다. 특히, 기술은 셀 라인 NHDF, 건강 한 개인의 방사선 x 선 세포와 CD34 + 세포의 급성 골수성 백혈병을 가진 환자에서의 정상적인 fibroblasts에 적용 됩니다. 방법의 세부 정보는 제시 결과의 맥락에서 지적 했다.

<table><tbody><tr><td>RPMI medium</td><td>Sigma-Aldrich</td><td>R0883</td><td>Medium for cell culture</td></tr><tr><td>Heparin sodium</td><td>ratiopharm</td><td>PZN 3029843</td><td>&amp;nbsp;Heparin 5,000 I.U. / mL</td></tr><tr><td>Sodium chloride solution 0.9%</td><td>B. Braun</td><td>PZN 1957154</td><td>Component of the anticoagulant stock solution</td></tr><tr><td>Ficoll-Paque Premium</td><td>GE Healthcare</td><td>17-5442-02</td><td>Medium for isolation of mononuclear cells</td></tr><tr><td>Trypsin solution 10X</td><td>Sigma-Aldrich</td><td>59427C</td><td>Enzyme for dissociation of fibroblasts in cell culture</td></tr><tr><td>CD34 MicroBead Kit</td><td>Miltenyi Biotec</td><td>130-046-702</td><td>Isolation of CD34+ myeloid progenitor cells</td></tr><tr><td>Diagnostic microscope slides</td><td>Thermo Scientific</td><td>ER-203B-CE24</td><td>Microscope slides</td></tr><tr><td>Megafuge 1.0 R</td><td>Heraeus</td><td>75003060</td><td>&amp;nbsp;Tabletop centrifuge&amp;nbsp;</td></tr><tr><td>Cytospin device</td><td></td><td></td><td></td></tr><tr><td>Lid</td><td>Heraeus</td><td>76003422</td><td>Lid for working without micro-tubes</td></tr><tr><td>Cyto container</td><td>Heraeus</td><td>75003416</td><td>Cyto container with 2 conical bores</td></tr><tr><td>Clip carrier</td><td>Heraeus</td><td>75003414</td><td>Carrier for holding a cyto container and a slide</td></tr><tr><td>Support insert</td><td>Heraeus</td><td>75003417</td><td>Support insert for holding a clip carrier</td></tr><tr><td>Triton X-100 (Octoxinol 9)</td><td>Thermo Scientific</td><td>85112</td><td>Detergent for permeabilization of cell membranes</td></tr><tr><td>Potassium hydroxide solution 1M</td><td>Merck Millipore</td><td>109107</td><td>Necessary for preparing the paraformaldehyde solution</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Fixation agent</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>Balanced salt solution</td></tr><tr><td>Chemiblocker</td><td>Merck Millipore</td><td>2170</td><td>Blocking agent</td></tr><tr><td>Mouse monoclonal anti-&amp;gamma;H2AX antibody (JBW301)</td><td>Merck Millipore</td><td>05-636</td><td>Primary antibody for detection of &amp;gamma;H2AX</td></tr><tr><td>Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td><td>Novus Biologicals</td><td>NB100-304</td><td>Primary antibody for detection of 53BP1</td></tr><tr><td>Alexa Fluor 488-conjugated goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11001</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 555-conjugated donkey anti-rabbit antibody</td><td>Invitrogen</td><td>A-31572</td><td>Secondary antibody</td></tr><tr><td>Vectashield mounting medium</td><td>Vector Laboratories</td><td>H-1200</td><td>Contains DAPI for staining of DNA</td></tr><tr><td>Axio Scope.A1</td><td>Zeiss</td><td>490035</td><td>Fluorescence microscope</td></tr><tr><td>Cool Cube 1 CCD camera</td><td>Metasystems</td><td>H-0310-010-MS</td><td>Camera system for digital recording</td></tr><tr><td>Isis software</td><td>Metasystems</td><td>Not applicable</td><td>Microscope software</td></tr></tbody></table>

immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks

DNA 이중 가닥 나누기 (DSB)는 심각한 DNA 병 변. 형성 및 DSB의 수리 분석은 게놈 무결성, genotoxicity, 방사선 생물학, 노화, 암, 및 신약 개발 등 연구 분야의 넓은 스펙트럼에 관련. 응답에서 DSB, 히스톤 H2AX 면역 형광 검사 현미경 검사 법에 의해 개별 핵 foci 감지를 형성 하는 여러 megabase 쌍의 영역에 떠들고 139에 phosphorylated은. 또한, 53BP1 (p53 바인딩 단백질 1)은 또 다른 중요 한 DSB 반응 단백질 nonhomologous 끝 동종 재결합 하면서 가입 하 여 DSB의 복구를 추진. ΓH2AX와 53BP1의 특정 기능에 따라 γH2AX 및 53BP1 면역 형광 검사 현미경 검사 법에 의해 결합 된 분석 DSB의 상세한 분석에 대 한 합리적인 접근 있을 수 있습니다. 이 원고는 단계별 프로토콜을 기술을 수행 하기 위한 조직적 노트와 보충을 제공 한다. 특히, γH2AX foci 패턴에 세포 주기의 영향 셀 라인 NHDF의 정상적인 fibroblasts에 시연입니다. 또한, biomarker는으로 γH2AX foci의 값은 건강 한 개인의 방사선 x 선 세포에 그려져 있습니다. 마지막으로, 유전자 불안정성의 γH2AX 및 53BP1 면역 형광 검사 현미경 검사 법에 의해 CD34 + 세포의 급성 골수성 백혈병을 가진 환자에서 조사.

셀에 γH2AX foci의 분석 G0/G1 단계 그리고 G2 단계 γH2AX foci (그림 5A) 고유한 형광 점으로 표시 하는 때에 가장 정확 하다. 반면, S 단계 세포에 γH2AX foci의 분석 복제 프로세스 (그림 5B)에 의해 발생 하는 분산된 팬 핵 γH2AX 얼룩에 의해 복잡 합니다.
셀의 고정 4% 수행한 PFA (10 분) 및 0.1% pe...

Watch this Scientific Journal Video about Immunofluorescence Microscopy of Î³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

이 원고는 γH2AX와 53BP1의 면역 형광 검사 현미경 검사 법에 의해 DNA 두 배 물가 틈의 분석을 위한 프로토콜을 제공합니다.

ΓH2AX와 53BP1 형성 및 DNA 두 배 물가 틈의 수리 분석에 대 한 면역 형광 검사 현미경 검사 법

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

immunofluorescence-microscopy-h2ax-53bp1-for-analyzing-formation

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Cancer Research

16.1K 조회수.  Medical Faculty Mannheim of the Heidelberg University. 이 원고는 γH2AX와 53BP1의 면역 형광 검사 현미경 검사 법에 의해 DNA 두 배 물가 틈의 분석을 위한 프로토콜을 제공합니다.

비디오: Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

면역 형광 검사 현미경 검사 법에 의해 변이 및 긴-지속 이중 가닥 DNA 틈에 DNA 복구 과정을 특성화

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

연구

암 연구학

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

인간 결장 암 세포에 있는 DNA 손상 및 복구 Foci의 면역 형광 화상 진찰

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

인간 말초 혈액 림프구의 γ-H2AX 포시 분석에 대한 자동화된 현미경 채점 방법

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

인간 말초 림프구에서 γH2AX 및 53BP1의 이중 면역형광

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

유전학

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

생물학

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

조직 샘플에서 γH2AX Foci의 Quantitation

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

면역형광에 의한 DNA 수리 단백질 상호작용의 시각화

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

<meta charset="utf8"></head><body>DNA 손상에서 단백질 상호 작용 및 국소화 평가

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>저자 스포트라이트: DNA 손상 반응에서 단백질 상호 작용을 특성화하기 위해 근접 리간드 분석과 Gamma-H2AX 염색의 결합

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

DNA 손상 부위의 단백질을 국소화하기 위한 Proximity Ligand Assay

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

ΓH2AX와 53BP1 형성 및 DNA 두 배 물가 틈의 수리 분석에 대 한 면역 형광 검사 현미경 검사 법

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면역 형광 검사 현미경 검사 법에 의해 변이 및 긴-지속 이중 가닥 DNA 틈에 DNA 복구 과정을 특성화

인간 결장 암 세포에 있는 DNA 손상 및 복구 Foci의 면역 형광 화상 진찰

인간 말초 혈액 림프구의 γ-H2AX 포시 분석에 대한 자동화된 현미경 채점 방법

인간 말초 림프구에서 γH2AX 및 53BP1의 이중 면역형광

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

조직 샘플에서 γH2AX Foci의 Quantitation

이온화 방사선 다음 γH2AX의 공간적 분포 평가

면역형광에 의한 DNA 수리 단백질 상호작용의 시각화

DNA 손상 부위의 단백질을 국소화하기 위한 Proximity Ligand Assay

DNA 손상 부위의 단백질을 국소화하기 위한 Proximity Ligand Assay