Name: immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks
Uploaded: 2017-11-03T14:00:00
Description: 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

Proje Alman Jose Carreras lösemi Vakfı (DJCLS 14 R/2017) tarafından desteklenmiştir.

Tüm yöntem tanımlamak burada Heidelberg Üniversitesi Tıp Fakültesi Mannheim Etik Komitesi II tarafından onaylanmıştır. Aydınlatılmış onam tüm bireyler elde edilen yazılı.
 1. hazırlık malzemeleri <ol><li>antikoagülan hisse senedi çözüm: mL % 0,9 Sodyum Klorür başına 200 IU heparin bir antikoagülan hisse senedi çözüm hazırlamak. Her koleksiyon tüpleri doldurun (birim çizmek 9 mL) 2 mL kan veya kemik iliği örnekleri geri çekilmesi önce ant...

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ΓH2AX ve 53BP1 ayirt mikroskobu oluşumu ve DSB tamiri araştırma konuları geniş bir yelpazede yapılan analiz etmek için kullanışlı bir yöntemdir. Deneyler sonucu etkileyen kritik hücre döngüsünün fiksasyon ve permeabilization hücrelerinin antikor ve donanım seçimi ve floresan mikroskop bilgisayar yazılımı için kullanılan ajanlar aşamasında parametreleridir.
Hücre döngüsünün etkisi γH2AX foci örgülerine katlanarak büyüyen hücre kültürünü NHDF normal fibr...

DNA sürekli zarar görmüş endojen tarafından (örneğin, çoğaltma stres, Reaktif oksijen türleri, DNA'ın iç istikrarsızlık) ve eksojen (örneğin, kimyasal radikaller, ışınlama) (Resim 1)1,2 kaynakları ,3,4. DNA hasarı arasında DNA çift iplikli kırılmalara (DSB) özellikle ciddi lezyonlardir ve hücre ölümü veya karsinojenezis neden olabilir. Yaklaşık 50 DSB hücre ve hücre döngüsü5başına ortaya çıkabilir. Memeli hücrelerinde homolog rekombinasyon (İK) ve nonhomologous sonu-katılma (NHEJ) DSB (Şekil 2) tamiri için büyük yollar olarak geliştirilmiştir. Saat geç S/G2 aşamasında oluşur ve sağlam bir kız kardeşi kullanır Kromatit potansiyel olarak hatasız onarım için bir şablon olarak. Baz çifti eklendiğinde veya kırık uçları ligasyonu önce rezeke karşılık, hücre döngüsü boyunca aktif ve potansiyel olarak mutajenik NHEJ. Buna ek olarak, alternatif son katılma NHEJ eksikliği6,7durumunda yavaş mutajenik yedekleme onarım mekanizması olarak meşgul olabilir.
DSB fosforilasyon Histon H2AX serin 139 birkaç megabase çiftleri her DSB çevresinde bir bölgede, bir neden. Şekillendirme nükleer resimde γH2AX foci adlı ve ayirt mikroskobu8tarafından algılanabilir. ΓH2AX daha fazla DSB-duyarlı protein alımı teşvik ve Kromatin remodeling, DNA tamiri ve sinyal iletim9ilgilenmektedir. Her γH2AX odak tek bir DSB temsil etmek için kabul edilir gibi DSB miktar ayirt mikroskobu tarafından hangi kanser hücre satırları ve hasta örnekleri10,11, göstermiş olduğu, mümkün 12 , 13 , 14. 53BP1 (p53 bağlayıcı protein 1) DSB onarım arabuluculuk içinde başka bir anahtar protein olduğunu. DSB-duyarlı proteinler, denetim noktası sinyal ve DSB biter15synapsis istihdamı ilgilenmektedir. Buna ek olarak, 53BP1 DSB onarım yolu seçiminde kritik bir rol oynamaktadır. İK EngellediBRÜKSEL16iken NHEJ doğru DSB tamiri tetikler. ΓH2AX ve 53BP1 DSB tamir hakiki fonksiyonları göz önüne alındığında, γH2AX ve 53BP1 ayirt mikroskobu tarafından eş zamanlı analiz oluşumu ve DSB tamiri kesin çözümleme için yararlı bir yöntem olabilir.
Bu el yazması ayirt mikroskobu, γH2AX ve 53BP1 hücre çekirdeği içinde gerçekleştirmek için adım adım bir protokol sağlar. Özellikle, teknik hücre kültürünü NHDF, x-ışını ışınlanmış lenfositler sağlıklı bir bireyin ve CD34 + bir hastada akut myeloid lösemi hücrelerinin normal fibroblastlar uygulanır. Yöntem ayrıntıları sunulan sonuçları bağlamında dikkat çekti.

<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 çift iplikli kırılmalara (DSB) ciddi DNA lezyonlardir. Oluşumu ve DSB tamiri analizi araştırma alanlarına genom bütünlük, genotoxicity, radyasyon biyolojisi, yaşlanma, kanser ve ilaç geliştirme de dahil olmak üzere geniş bir yelpazede uygun olur. DSB yanıt olarak, Histon H2AX serin 139 ayrı nükleer foci tespit şekillendirme birkaç megabase çiftleri bir bölgede, ayirt mikroskobu tarafından fosforile. Buna ek olarak, 53BP1 (p53 bağlayıcı protein 1) başka bir önemli DSB-duyarlı protein DSB tamiri katılarak nonhomologous sonu-homolog rekombinasyon önlenmesi ise teşvik olduğunu. ΓH2AX ve 53BP1 belirli fonksiyonları göre γH2AX ve 53BP1 ayirt mikroskobu ile kombine analizi DSB ayrıntılı bir analiz için makul bir yaklaşım olabilir. Bu el yazması teknik gerçekleştirmek için metodik notları ile desteklenmiş bir adım adım iletişim kuralı sağlar. Özellikle, hücre döngüsünün etkisi γH2AX foci örgülerine hücre kültürünü NHDF normal fibroblastlar içinde gösterilmiştir. Ayrıca, γH2AX resimde değeri bir biyomarker olarak sağlıklı bir bireyin ışınlanmış x-ray lenfosit tasvir edilir. Son olarak, genetik istikrarsızlık tarafından γH2AX ve 53BP1 ayirt mikroskobu hücrelerdeki CD34 + olan bir hastanın akut myeloid lösemi soruşturma.

ΓH2AX foci hücrelerdeki G0/G1 evresi ve ne zaman γH2AX foci farklı floresan noktalar (5A rakam) görünür G2 faz en doğru analizidir. Buna ek olarak, γH2AX foci S aşamasında hücrelerdeki analizi (şekil 5B) çoğaltma işlemi tarafından neden dağınık pan-nükleer γH2AX speckles tarafından karmaşıktır.
Hücreleri fiksasyonu %4 ile gerçekleştirilen PF...

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 ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

Bu el yazması bir iletişim kuralı tarafından γH2AX ve 53BP1 ayirt mikroskobu DNA çift iplikli kırılmalara analiz için sağlar.

ΓH2AX ve 53BP1 oluşumu ve DNA çift iplikli kırılmalara tamiri analiz etmek için ayirt mikroskobu

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 ...

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Cancer Research

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

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Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment (Video) | JoVE 

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