원자력 과학 및 공학의 호주 연구소의 지원 인정받고 있습니다. TCK는 AINSE 보너스받는했다. Epigenomic 의학 연구소는 국민 건강과 호주의 의료 연구위원회 (566559)에 의해 지원됩니다. LM은 멜버른 연구 (멜버른 대학)와 바이오 메디컬 이미징 CRC의 보조 장학금으로 지원됩니다. 모나 마이크로 이미징의 지원 (DRS 스티븐 코디 Iśka 카마 이클)이 작품 소중한했다.

<html> 셀 준비 <ol><li> 37, 표피 성장 인자, 소 뇌하수체 추출물 및 20 μg / ML gentamicin과 보충 ° C와 5% CO 2; 인간 keratinocytes (FEP - 1811)은 (Invitrogen K - SFM) Keratinocyte - 세럼 무료 매체 성장했다. </li><li> 단일 세포 현탁액은 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) (0.05 % V / V)로 분리하여 준비했습니다 </li><li> 전지는 8 잘 연구실 테크 II microchamber 슬라이드에서 씨앗을 품고 있었다 (10,000 셀 / 음)과 슬라이드가 37 ° C와 5 % CO 2에서 3 일간 incubated되었습니다. </li></ol> 조사 <ol><li> 세포는 137 고사 소스를 (; Nordion 국제, ON, 캐나다, 20.6 초 / 쥐 Gammacell 1000 엘리트 irradiator)를 사용하여이 쥐와 함께 얼음에 조사되었다 </li><li> Unirradiated 제어 및 2 쥐 조사 전지는 37 ° C와 5% CO 2에서 1 시간 동안 incubated되었습니다. </li></ol> Immunofluorescence 염색법 <ol><li> 미디어는 밀고되어 세포가 잘 당 PBS의 300μl (W / O 칼슘 2 + 또는 MG 2 +)로 씻어 5 분 동안 궤도 믹서에 회전 하였다. </li><li> 버퍼는 밀고와 paraformaldehyde 각 잘 추가되었고 슬라이드가 10 분 동안 상온에서 incubated 있었다 신선한 4퍼센트 (V / V)의 100μl했습니다. 모든 incubations은 humidified 얼룩 트러프에서 수행되었다 </li><li> 전지는 다음 PBS (W / O 칼슘 2 + 또는 MG 2 +)로 세탁했다. 슬라이드가 5 분 궤도 믹서에 코플린 병 및 회전에 배치했다. 이 세척 단계는 더 이상 두 번 반복되었다. </li><li> 버퍼 밀고했고 초과 PBS는 부드럽게 blotted되었습니다. </li><li> 세포 100ml 트리톤 X - 100 (0.1 % V / V) 당 잘하고 실온에서 10 분 배양을 사용하여 permeabilized되었습니다. </li><li> 세포 PBS로 씻은했다 (W / O 칼슘 2 + 또는 MG 2 +)와 같은 3 단계 이상 4 설명했다. </li><li> 비 - 특정 단백질 바인딩은 상온에서 잘하고 이십분 부화 당 BSA의 100ml (1 % V / V)로 차단되었습니다. </li><li> 초과 BSA는 밀고되었으며 기본 마우스 단클론 항 phospho - H2AX 히스톤 항체 (1 % BSA에 1:500 희석, Millipore)의 100μl는 상온에서 1 시간 배양 각 잘 추가되었습니다. </li><li> 세포 PBS로 씻은했다 (W / O 칼슘 2 + 또는 MG 2 +) 위의 3 단계와 4 단계에서 설명한 및 보조 항체의 100μl (알렉사 플루어 488 염소 방지 마우스 IgG와 incubated 1 % BSA에 1:500 희석 ; Invitrogen) 어둠 속에서 실온에서 45 분간 잘마다. (희석 항체가 절차를 통해 어둠에 보관되었습니다) </li><li> 세포 PBS로 씻은했다 (W / O 칼슘 2 + 또는 MG 2 +)와 같은 3 단계 이상 4 설명했다. 그러나, 빛에 노출 호일을 사용하여 최소화했다. </li><li> 잘 따라와 실온에서 10 분 배양, 핵 counterstaining은 100ml TOPRO3 (Invitrogen 1:500 희석)으로 수행되었습니다 </li><li> 전지는 PBS (W / O 칼슘 2 + 또는 MG 2 +)가로 씻은 것처럼 위의 단계 10 설명했다. </li><li> 챔버 스는 조심스럽게 슬라이드에서 제거되었습니다, 과잉 수분이 blotted되었고 슬라이드는 건조 공기를 허용되었다. </li><li> GOLD 방지 퇴색 솔루션 (Invitrogen)를 연장 중 하나는 방울마다 잘 추가되었습니다과 슬라이드가 탑재되었다 (22x50 mm coverslip) 및 슬라이드의 가장자리 주위에 초과 액체는 bl​​otted되었습니다. </li><li> 슬라이드는 매니큐어로 밀봉하기 전에 실온에서 추가로 30 분 어둠에 보관했다. </li><li> 슬라이드 4에서 하룻밤 저장된 ° C 분석하기 전에 어둠 속이라서. </li></ol> 현미경 / 분석 <ol><li> (- 알렉사 플루어 488 염소 안티 - 마우스 IgG γH2AX)과 멀리 붉은 레이저 (TOPRO - 3) 자이스 혈구 LSM510 메타 공촛점 Microscpe은 표준 GFP를 사용하여 이미지를 획득하는 데 사용됩니다. 일반적으로 63 X 오일 침지 객관적인 렌즈가 사용됩니다. 이미지는 0.5 μm의의 스텝 크기와 Z - 시리즈 패턴에 인수됩니다. 0.5 μm의의 단계 크기는 핵에서 다른 비행기에 foci 선물의 손실을 최소화하기 위해 선정되었습니다. 분석하는 동안, 개인 비행기는 deconvoluted 및 foci의 중복 (톱 - 모자 필터 적용)를 최소화하기 위해 최대 예상 이미지를 생산하기 위해 정렬됩니다. </li><li> Metamorph (분자 디바이스, 미국)는 foci의 개수를 분석하는 데 사용되었다. 임계값이 배경을 제외 적용된 후이 프로그램은 각 셀에 foci의 수를 quantitates. 이 정보는 추가 분석을 위해 Microsoft Excel 스프레드 시트에 기록됩니다. </li></ol><img src="/files/ftp_upload/1957/1957fig1.jpg" alt="그림 1" /> 그림 1. 치료 인간 keratinocytes에서 2 쥐와 조사 37에서 추가로 1 시간 동안 incubated 세포 γH2AX foci (녹색)의 Immunofluorescence 시각화 ° C 5 % CO 2. DNA는 TOPRO - 3 (블루) 물들일했습니다. 이미지 자이스 혈구 LSM 510 메타 공촛점 현미경을 사용하여 인수했다. 바 = 10 μm의. e_content &quot;&gt; <img src="/files/ftp_upload/1957/1957fig2.jpg" alt="그림 2" /> 그림 2. 인간 keratinocytes에서 2 쥐와 조사 37에서 추가로 1 시간 동안 incubated 세포 γH2AX foci (녹색)의 Immunofluorescence 시각화 ° C 5 % CO 2. 이미지는 모든 foci가 인수되었습니다 보장하기 위해 0.5 μm의 Z - sectioning (1-9)를 사용하여 자이스 혈구 LSM 510 메타 공촛점 현미경을 사용하여 인수했다. 이미지는 다음 Metamorph를 사용하여 quantitation 위해 쌓아했다. DNA는 TOPRO - 3 (블루) 물들일했습니다. 스택 γH2AX 및 파랑 이미지는 시각화를 위해 쌓아되었습니다. 바 = 10 μm의.

<ol>
	<li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+H2AX+and+cancer.">Gamma H2AX and cancer.</a> Nature Reviews Cancer. 8 (12), 957-967 (2008).</li><li>Savic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Dynamic+[gamma]-H2AX+Domains+along+Broken+DNA+Strands+Is+Distinctly+Regulated+by+ATM+and+MDC1+and+Dependent+upon+H2AX+Densities+in+Chromatin.">Formation of Dynamic [gamma]-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin.</a> Molecular Cell. 34 (3), 298-310 (2009).</li><li>Downs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+chromatin-modifying+activities+to+phosphorylated+histone+H2A+at+DNA+damage+sites.">Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites.</a> Mol Cell. 16 (6), 979-990 (2004).</li><li>Chowdhury, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[gamma]-H2AX+Dephosphorylation+by+Protein+Phosphatase+2A+Facilitates+DNA+Double-Strand+Break+Repair.">[gamma]-H2AX Dephosphorylation by Protein Phosphatase 2A Facilitates DNA Double-Strand Break Repair.</a> Molecular Cell. 20 (5), 801-809 (2005).</li><li>Nakada, S., Chen, G., Gingras, A., Durocher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PP4+is+a+gamma+H2AX+phosphatase+required+for+recovery+from+the+DNA+damage+checkpoint.">PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint.</a> EMBO Rep. 9 (10), 1019-1026 (2008).</li><li>Altaf, M., Auger, A., Covic, M., C&#244;t&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connection+between+histone+H2A+variants+and+chromatin+remodeling+complexes.">Connection between histone H2A variants and chromatin remodeling complexes.</a> Biochem Cell Biol. 87 (1), 35-50 (2009).</li><li>Kusch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+by+Tip60+Is+Required+for+Selective+Histone+Variant+Exchange+at+DNA+Lesions.">Acetylation by Tip60 Is Required for Selective Histone Variant Exchange at DNA Lesions.</a> Science. 306, 2084-2087 (2004).</li><li>Hurlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88 (2), 570-574 (1991).</li><li>Dickey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. , (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이온화 방사선 (γ 선), 빠르게 γH2AX foci 양식 및 foci 번호를 다음 노출 30~60분 2 사이 최대에 도달. 따라서, 우리 1시간 후 방사선 시점은 초기 DSB 형성을 반영합니다. 우리는 우리의 실험 2 쥐의 임상 관련 방사선 선량을 사용했습니다. 그러나 방법은 초기 DSB 형성의 검출 4 쥐 위해 방사선 접종에 사용할 수있는, foci 상당한 중복 높은 복용에 정확한 quantitation 걸로. γH2AX foci가 같지는 숫자 결과, 수리​​로 인한 손실로 높은 방사선 접종은 더 후 방사선 배양 시간에 사용할 수 있습니다. 일반적으로, 최대 24 시간 후 방사선 배양 시간 4시간하며 모니터링 DNA의 수리를 위해 사용됩니다. 우리는 DNA가 우리 공촛점 현미경의 여기 레이저의 제한 TOPRO - 3으로 인해 스테인 사용하고 있습니다. 더 일반적으로, 4,6 - diamidino - 2 - phenylindole의 dihydrochloride (DAPI)는 핵 counterstaining에 사용됩니다. 우리가 공촛점 현미경과 epifluorescent 현미경 사용되지만 Z를 sectioning 용량 것이 적절합니다. immunofluorescence 방법은 다른 자기편 암 및 정상 세포 라인에 적합합니다, 우리는 인간 glioblastoma 정상적인 인간의 내피 세포와 쥐의 배아 H9c2 심실 myocytes을 T98G 테스트했습니다. 마지막으로, γH2AX의 quantitation 우리 실험 및 수리 (방사선 감도)에 의해 그림과 같이 모니터링 DNA 손상에 대한 방사선 유발 DNA 손상을 이온화의 맥락에서 유용합니다. 분석은 방사선 (즉, 방사선 수호자와 sensitizers)에 세포 반응을 조절 물질의 효능을 평가에도 유용합니다. 또한, γH2AX 암 10 주로, 노화와 질병의 잠재적인 분자 마커로 대두되고 있습니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Keratinocyte-Serum Free Medium (K-SFM)</td>
<td>Media</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Keratinocyte media supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE).</td>
</tr>
<tr>
<td>Lab Tek lI Chamber Slides (8-well)</td>
<td>Chamber Slides</td>
<td>Nunc Denmark</td>
<td>NUN154534</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>Coverslips</td>
<td>Menzel-Gl&auml;ser</td>
<td>CS2250100</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin (BSA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A7906</td>
<td>BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.</td>
</tr>
<tr>
<td>PBS (without Ca2+ and Mg2+)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17-517Q</td>
<td>&nbsp;</td>
<tr>
<td>0.5% Trypsin-EDTA x10</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>Trypsin-EDTA (0.05%) used to detach cells from culture flasks.</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>Triton X-100 (0.1%) used to permeabilise cells.</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>158127</td>
<td>Paraformaldehyde (4%) used to fix cells.</td>
</tr>
<tr>
<td>Mouse monoclonal anti-phospho histone-H2AX antibody</td>
<td>Primary Antibody</td>
<td>Millipore</td>
<td>16193</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen</td>
<td>11029</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>TOPRO3</td>
<td>DNA Stain</td>
<td>Invitrogen</td>
<td>T3605</td>
<td>DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.</td>
</tr>
<tr>
<td>ProLong Gold</td>
<td>Anti-fade solution</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>Refractive index of 1.42 at 20&deg;C.</td>
</tr>
<tr>
<td>Tissue Culture Flask, Vented Cap</td>
<td>Culture Flask</td>
<td>BD Falcon</td>
<td>353112</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Dish (150x25mm)</td>
<td>Petridish</td>
<td>BD Falcon</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>Coplin Jar, glass</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>1771-OG</td>
<td>&nbsp;</td>
<tr>
<td>Staining Trough</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>V1991.99</td>
<td>&nbsp;</td>
<tr>
<td>Gammacell 1000 Elite Irradiator</td>
<td>Gamma Irradiator</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss LSM 510 Meta Confocal</td>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantification of &gamma;h2ax foci in response to ionising radiation

내생 신진 대사 과정 중 또는 외인성 소스에 의해 유도된 아르 DNA 이중 가닥 나누기 (DSBs)는, 게놈 무결성 생존과 보존에 관한 가장 중요한 DNA의 병변 중 하나입니다. DSBs의 유도에 초기 반응은 γH2AX을 형성, 높​​은 보존 C - 터미널 SQEY의 모티브에서 세린 - 139 잔여물에서 H2A 히스톤 변형, H2AX의 인산화입니다<sup&gt; 1</sup&gt;. DSBs의 유도에 따라, H2AX는 빠르게 phosphatidyl - inosito 3 키나제 (PIKK) 단백질의 가족 운동 실조증 telangiectasia 변이된 (ATM), DNA - 단백질 키나제 촉매 subunit와 ATM과 RAD3 관련하여 phosphorylated입니다 (ATR)<sup&gt; 2</sup&gt;. 일반적으로 단지 몇베이스 쌍 (BP)은 DNA 손상 신호 및 수리에 염색질 수정의 중요성을 감안할 때, 그러나, 중요한 신호 증폭이, DSB에 연루됩니다. H2AX의 인산화는 DSB 당 전체 세포 H2AX의 약 0.03 %에 영향을 미치는, 염색질의 인접 지역에 ATM 확산에 의해 주로 중재<sup&gt; 2,3</sup&gt;. 이것은 쉽게 시각과 immunofluorescence 현미경에 의해 quantitated 수있는 DSB와 이산 γH2AX foci 형성의 결과의 사이트를 둘러싼 염색질의 ~ 2 MBP 영역을 걸쳐 약 2000 H2AX 분자의 인산화에 해당하는<sup&gt; 2</sup&gt;. DSB에 γH2AX의 손실 복구를 반영하지만, DSBs의 완전한 복구를 정의하는 무엇 같은 논쟁이있다, 그것은 DNA의 양쪽 가닥이 복귀하는 것은 그러나 적절한 것을 제안되었습니다, 그것은 또한 제안되었음을에 다시 instatement 압축의 원래 염색질 상태가 필요합니다<sup&gt; 4-8</sup&gt;. γH2AX의 disappearence 적어도 부분적으로 포함, phosphatases, 인산 가수 분해 효소 2A 및 인산 가수 분해 효소 4C에 의해 dephosphorylation<sup&gt; 5,6</sup&gt;. 또한, H2A.Z와 히스톤 교류를 포함한 재배포하여 γH2AX의 제거가 연루되어있다<sup&gt; 7,8</sup&gt;. 중요한 것은 γH2AX foci의 정량 분석​​은 의료 및 핵 연구에 응용 프로그램의 다양한되었다. 여기서는 γ - 반구 자기편 인간 keratinocytes에서 γH2AX foci의 탐지 및 quantitation에 의해 초기의 DNA 손상 평가를 위해 가장 일반적으로 사용되는 방법을 보여주 immunofluorescence<sup&gt; 9</sup&gt;.

Watch this Scientific Journal Video about Quantification of ÃŽÂ³H2AX Foci in Response to Ionising Radiation at JoVE.com

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

분자 마커로 γH2AX 형성을 사용하여 DNA 이중 스트랜드 줄무늬의 부량 방사선 생물학에서 매우 중요한 도구가되었습니다. 여기 우리는 방사선에 노출 후 세포의 γH2AX foci의 부량위한 immunofluorescence 분석의 사용을 보여줍니다.

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 ...

quantification-of-h2ax-foci-in-response-to-ionising-radiation

Nanyang Technological University

Research

JoVE Journal

Biology

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

20.3K 조회수.  The Alfred Medical Research and Education Precinct. 분자 마커로 γH2AX 형성을 사용하여 DNA 이중 스트랜드 줄무늬의 부량 방사선 생물학에서 매우 중요한 도구가되었습니다. 여기 우리는 방사선에 노출 후 세포의 γH2AX foci의 부량위한 immunofluorescence 분석의 사용을 보여줍니다.

비디오: Quantification of γH2AX Foci in Response to Ionising Radiation

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) ...

연구

생물학

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 ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

의 형광 단백질의 편광 Translocation XenopusWnt 신호에 응답> Ectoderm

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA의 손상 응답 단백질의 2와 3 차원 라이브 셀 이미징

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

구강 안면 표현형의 정량화에<em&gt; Xenopus의</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

히드라의 글루타티온 유도 먹이 반응을 측정

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

내피 세포 접착 정량<em&gt; 체외</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

실험 접근법은 미토콘드리아 현지화 및 핵 세포주기 키나제,는 Cdk1의 기능을 연구하는

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

포유 동물 세포에서 초기 DNA에 바인딩 단백질의 시험 BrdU의 칩 슬롯 서양 기술을 사용하여

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...