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>

<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 border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">RPMI medium</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">R0883</td>
			<td style="width:200px;">Medium for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Heparin sodium</td>
			<td style="width:119px;">ratiopharm</td>
			<td style="width:104px;">PZN 3029843</td>
			<td style="width:200px;">&#160;Heparin 5,000 I.U. / mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium chloride solution 0.9%</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:104px;">PZN 1957154</td>
			<td style="width:200px;">Component of the anticoagulant stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Ficoll-Paque Premium</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:104px;">17-5442-02</td>
			<td style="width:200px;">Medium for isolation of mononuclear cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Trypsin solution 10X</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">59427C</td>
			<td style="width:200px;">Enzyme for dissociation of fibroblasts in cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">CD34 MicroBead Kit</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:104px;">130-046-702</td>
			<td style="width:200px;">Isolation of CD34+ myeloid progenitor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Diagnostic microscope slides</td>
			<td>Thermo Scientific</td>
			<td>ER-203B-CE24</td>
			<td style="width:200px;">Microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Megafuge 1.0 R</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003060</td>
			<td style="width:200px;">&#160;Tabletop centrifuge&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Cytospin device</td>
			<td style="width:119px;"></td>
			<td style="width:104px;"></td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Lid</td>
			<td style="width:119px;">Heraeus</td>
			<td>76003422</td>
			<td style="width:200px;">Lid for working without micro-tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cyto container</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003416</td>
			<td style="width:200px;">Cyto container with 2 conical bores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Clip carrier</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003414</td>
			<td style="width:200px;">Carrier for holding a cyto container and a slide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Support insert</td>
			<td style="width:119px;">Heraeus</td>
			<td style="width:104px;">75003417</td>
			<td style="width:200px;">Support insert for holding a clip carrier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Triton X-100 (Octoxinol 9)</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:104px;">85112</td>
			<td style="width:200px;">Detergent for permeabilization of cell membranes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Potassium hydroxide solution 1M</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">109107</td>
			<td style="width:200px;">Necessary for preparing the paraformaldehyde solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Paraformaldehyde</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">P6148</td>
			<td style="width:200px;">Fixation agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Phosphate buffered saline</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:104px;">D8537</td>
			<td style="width:200px;">Balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Chemiblocker</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">2170</td>
			<td style="width:200px;">Blocking agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Mouse monoclonal anti-&#947;H2AX antibody (JBW301)</td>
			<td style="width:119px;">Merck Millipore</td>
			<td style="width:104px;">05-636</td>
			<td style="width:200px;">Primary antibody for detection of &#947;H2AX</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td>
			<td style="width:119px;">Novus Biologicals</td>
			<td style="width:104px;">NB100-304</td>
			<td style="width:200px;">Primary antibody for detection of 53BP1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Alexa Fluor 488-conjugated goat anti-mouse antibody</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:104px;">A-11001</td>
			<td style="width:200px;">Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Alexa Fluor 555-conjugated goat anti-rabbit antibody</td>
			<td style="width:119px;">Invitrogen</td>
			<td style="width:104px;">A-21428</td>
			<td style="width:200px;">Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Vectashield mounting medium</td>
			<td style="width:119px;">Vector Laboratories</td>
			<td style="width:104px;">H-1200</td>
			<td style="width:200px;">Contains DAPI for staining of DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Axio Scope.A1</td>
			<td style="width:119px;">Zeiss</td>
			<td style="width:104px;">490035</td>
			<td style="width:200px;">Fluorescence microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Cool Cube 1 CCD camera</td>
			<td style="width:119px;">Metasystems</td>
			<td style="width:104px;">H-0310-010-MS</td>
			<td style="width:200px;">Camera system for digital recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Isis software</td>
			<td style="width:119px;">Metasystems</td>
			<td style="width:104px;">Not applicable</td>
			<td style="width:200px;">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 ÃŽÂ³H2AX 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

Research

JoVE Journal

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

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

단일의 검사 및 포유류 세포에서 이중 가닥 브레이크 수리에 대 한 레이저 마이크로-방사선의 응용

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

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Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

순환 종양 세포를 캡처에 대 한 의료 와이어를 사용 하 여 종양 세포의 특성: 면역 형광 DNA 물고기 기반으로 3 차원 접근

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

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 ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

환자 생체 유체에서 종양 관련 순환 DNA의 검출 및 모니터링

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

암 연구에서 치료 및 진단 항체 바이오 유통 평가를 위한 생체내 면역형광 국소화

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

표적 항원 특이적 치료 항체의 종양 및 조직 분포 분석

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

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 ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

암 환자의 혈장 샘플에서 세포없는 DNA 의 검출

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

생체 발광을 사용하여 마우스의 유방암 성장 및 전이성 콜로니 형성 모니터링

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.