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

Prosjektet ble støttet av tysk José Carreras leukemi stiftelsen (DJCLS 14 R/2017).

alle metodene som er beskrevet her er godkjent av etiske komiteen II av det medisinske fakultet Mannheim av Heidelberg University. Skriftlig samtykke ble innhentet av alle personer som.
 1. forberedelse for materiale <ol><li>antikoagulerende lagerløsning: forberede en antikoagulerende lager løsning av 200 IE heparin per mL i 0,9% natriumklorid. Fyll hver samling rør (tegne volum 9 mL) med 2 mL antikoagulerende lager løsningen før uttak av blod og benmarg prøvene.</li>...

<ol>
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Immunofluorescence mikroskopi γH2AX og 53BP1 er en nyttig metode for å analysere dannelse og reparasjon av DSB i et bredt spekter av forskningsfelt. Kritiske parametere som påvirker utfallet av eksperimentene er fasen av cellen syklus, agenter brukes til fiksering og permeabilization av celler, valg av antistoffer, og maskinvaren og programvaren til fluorescens mikroskopet.
Påvirkning av cellen syklus på γH2AX foci mønstre ble demonstrert av eksponensielt voksende normal fibroblaster av...

DNA er kontinuerlig skadet av endogene (f.eks, replikering stress, reaktive oksygen arter, iboende ustabilitet av DNA) og ytre (f.eks, kjemiske radikaler, bestråling) kilder (figur 1)1,2 ,3,4. Blant DNA skade, DNA dobbel-strand bryter (DSB) er spesielt alvorlige lesjoner og kan indusere celledød eller kreft. 50 DSB kan oppstå per celle og celle syklus5. I pattedyrceller, homologe rekombinasjon (HR) og nonhomologous slutten med (NHEJ) utviklet som store trasé for reparasjon av DSB (figur 2). HR forekommer i slutten S/G2 fase og bruker en intakt søster chromatid som en mal for potensielt feilfrie reparasjon. Til sammenligning er NHEJ aktiv hele cellen syklus og potensielt mutagene som base parene kan legges til eller kappet før ligation av ødelagt. I tillegg kan alternativ slutten-begynte være engasjert som en langsom mutagene sikkerhetskopiere reparasjon mekanisme ved NHEJ mangel6,7.
DSB induserer fosforylering av histone H2AX på Serine 139 i en region av flere megabase rundt hver DSB. Forming kjernefysiske fokuspunktene kalles γH2AX foci og er detectable av immunofluorescence mikroskopi8. ΓH2AX fremmer rekruttering av ytterligere DSB svarer proteiner og er involvert i chromatin remodeling, DNA-reparasjon og signal signaltransduksjon9. Som hver γH2AX fokus anses å representere en enkelt DSB, er kvantifisering av DSB av immunofluorescence mikroskopi mulig, som har vist i kreftcelle linjer og pasienten prøver10,11, 12 , 13 , 14. 53BP1 (p53 binding protein 1) er en annen viktig protein på formidling DSB reparasjon. Det er involvert i rekrutteringen av DSB svarer proteiner, checkpoint signalering og Adept DSB slutter15. I tillegg spiller 53BP1 en avgjørende rolle i DSB repair pathway valg. Det trigger reparasjon av DSB mot NHEJ HR er forhindret16. Vurderer ekte funksjonene γH2AX og 53BP1 i DSB reparasjon, kan samtidig analyse av γH2AX og 53BP1 av immunofluorescence mikroskopi være en nyttig metode for nøyaktig analyse av dannelse og reparasjon av DSB.
Dette manuskriptet gir en trinnvis protokoll for å utføre immunofluorescence mikroskopi γH2AX og 53BP1 i celle kjerner. Spesielt brukes teknikken i vanlig fibroblaster av cellen linjen NHDF, røntgen bestrålt lymfocytter av en frisk person og CD34 + celler i en pasient med akutt myelogen leukemi. Detaljer om metoden er påpekt i forbindelse med presenterte resultatene.

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	<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 dobbel-strand bryter (DSB) er alvorlig DNA lesjoner. Analyse av dannelse og reparasjon av DSB er relevant i et bredt spekter av forskningsfelt inkludert genomet integritet, gentoksisitet, stråling biologi, aldring, kreft og narkotika utvikling. Svar på DSB, er histone H2AX fosforylert på Serine 139 i en region av flere megabase par danner diskret kjernefysiske foci detectable av immunofluorescence mikroskopi. I tillegg er 53BP1 (p53 binding protein 1) er en annen viktig DSB svarer protein fremme reparasjon av DSB melder nonhomologous slutten-mens forebygge homologe rekombinasjon. Ifølge de bestemte funksjonene γH2AX og 53BP1, kan kombinerte analyse av γH2AX og 53BP1 av immunofluorescence mikroskopi være en fornuftig tilnærming for en detaljert analyse av DSB. Dette manuskriptet gir en trinnvis protokoll med metodisk notater for å utføre teknikken. Spesielt er påvirker celle syklusen γH2AX foci mønstre demonstrert i normal fibroblaster av cellen linjen NHDF. Videre er verdien av γH2AX fokuspunktene som en biomarkør avbildet i x-ray bestrålt lymfocytter av en frisk person. Til slutt, genetisk ustabilitet er undersøkt i CD34 + celler i en pasient med akutt myelogen leukemi av immunofluorescence mikroskopi γH2AX og 53BP1.

Analyse av γH2AX foci i celler er mest nøyaktige i fasen G0/G1 og G2 fasen når γH2AX foci vises som distinkte fluorescerende prikker (figur 5A). I kontrast, er analyse av γH2AX foci i celler i S fasen komplisert ved spredt pan-kjernefysiske γH2AX flekker forårsaket av replikeringsprosessen (figur 5B).
Fiksering av celler ble utført med 4% PFA (10 min) og permeab...

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

Dette manuskriptet gir en protokoll for analyse av DNA dobbel-strand bryter ved immunofluorescence mikroskopi γH2AX og 53BP1.

Immunofluorescence mikroskopi γH2AX og 53BP1 for å analysere dannelse og reparasjon av DNA dobbel-strand bryter

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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