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

Projektet blev støttet af den tyske José Carreras leukæmi Foundation (DJCLS 14 R/2017).

alle metoder beskrevet her er blevet godkendt af de etiske komité II af den medicinske fakultet Mannheim i Heidelberg Universitet. Skrevet blev indhentet informeret samtykke fra alle enkeltpersoner.
 1. forberedelse af materialer <ol><li>antikoagulans stamopløsning: forberede en antikoagulerende stamopløsning af 200 IE heparin pr. mL i 0,9% natriumchlorid. Fyld hver indsamling rør (tegne bind 9 mL) med 2 mL af den antikoagulerende stamopløsning før tilbagetrækning af...

<ol>
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K., 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=Evidence+for+ongoing+DNA+damage+in+multiple+myeloma+cells+as+revealed+by+constitutive+phosphorylation+of+H2AX.">Evidence for ongoing DNA damage in multiple myeloma cells as revealed by constitutive phosphorylation of H2AX.</a> Leukemia. 25 (8), 1344-1353 (2011).</li><li>Yu, T., MacPhail, S. H., Banath, J. P., Klokov, D., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+expression+of+phosphorylated+histone+H2AX+in+tumors+in+relation+to+DNA+double-strand+breaks+and+genomic+instability.">Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability.</a> DNA Repair (Amst). 5 (8), 935-946 (2006).</li><li>Sedelnikova, O. A., 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=GammaH2AX+in+cancer+cells:+a+potential+biomarker+for+cancer+diagnostics,+prediction+and+recurrence.">GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence.</a> Cell Cycle. 5 (24), 2909-2913 (2006).</li><li>Chen, E., 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=JAK2V617F+promotes+replication+fork+stalling+with+disease-restricted+impairment+of+the+intra-S+checkpoint+response.">JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response.</a> Proc Natl Acad Sci U S A. 111 (42), 15190-15195 (2014).</li><li>Popp, H. 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Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.</a> Scand J Clin Lab Invest Suppl. 97, 77-89 (1968).</li><li>Bain, B., Pshyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+reactivity+in+mixed+leukocyte+cultures+after+separation+of+mononuclear+cells+on+Ficoll-Hypaque.">Enhanced reactivity in mixed leukocyte cultures after separation of mononuclear cells on Ficoll-Hypaque.</a> Transplant Proc. 4 (2), 163-164 (1972).</li><li>Popp, H. 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Immunfluorescens mikroskopi af γH2AX og 53BP1 er en nyttig metode til at analysere dannelse og reparation af DSB i et bredt spektrum af forskningsområder. Kritiske parametre, der påvirker resultatet af forsøgene er fasen af cellecyklus, de midler, som anvendes til fiksering og permeabilization af celler, valget af antistoffer, og hardware og software af fluorescens mikroskop.
Indflydelse af cellecyklus på γH2AX foci mønstre blev demonstreret af eksponentielt voksende normale fibroblaste...

DNA er løbende beskadiget af endogene (fx, replikering stress, reaktive ilt arter, iboende ustabilitet af DNA) og eksogen (fx, kemiske radikaler, bestråling) kilder (figur 1)1,2 ,3,4. Blandt DNA-skader, DNA dobbelt-strenget pauser (DSB) er særligt alvorlige læsioner og kan fremkalde celledød eller carcinogenese. Omkring 50 DSB kan opstå pr. celle og cellecyklus5. I pattedyrceller homologe rekombination (HR) og nonhomologous ende-sammenføjning (NHEJ) udviklet som store veje for reparation af DSB (figur 2). HR opstår i slutningen S/G2 fase og bruger en intakt søster chromatid som en skabelon for potentielt fejlfri reparation. I sammenligning er NHEJ aktive i hele cellecyklus og potentielt mutagene som basepar kan være tilføjet eller resektion før ligatur af de brudte ender. Derudover kan alternative ende-sammenføjning være ansat som en langsom mutagene back-up reparation mekanisme i tilfælde af NHEJ mangel6,7.
DSB fremkalde fosforylering af Histon H2AX på Serin 139 i en region af flere megabase par omkring hver DSB. De danner nukleare foci er opkaldt γH2AX foci og påvises ved immunfluorescens mikroskopi8. ΓH2AX fremmer ansættelse af yderligere DSB-responderende proteiner og er involveret i kromatin remodeling, DNA reparation og signaltransduktion9. Som hver γH2AX fokus anses for at repræsentere et enkelt DSB, er kvantificering af DSB ved immunfluorescens mikroskopi mulige, som er blevet påvist i kræft cellelinjer og patient prøver10,11, 12 , 13 , 14. 53BP1 (p53 bindende protein 1) er en anden vigtig protein i mægle DSB reparation. Det er involveret i ansættelse af DSB-responderende proteiner, checkpoint signalering og synapsis DSB slutter15. Derudover spiller 53BP1 en afgørende rolle i DSB reparation pathway valg. Det udløser reparation af DSB mod NHEJ, mens HR er forhindret16. I betragtning af den ægte funktioner γH2AX og 53BP1 i DSB reparation, kan simultan analyse af γH2AX og 53BP1 ved immunfluorescens mikroskopi være en nyttig metode til præcis analyse af dannelse og reparation af DSB.
Dette håndskrift giver en trinvis protokol for at udføre immunofluorescens mikroskopi af γH2AX og 53BP1 i cellekerner. Specifikt, anvendes teknikken i normal fibroblaster af cellelinie NHDF, x-ray bestrålet lymfocytter af en sund person og CD34 + celler af en patient med akut myeloid leukæmi. Nærmere oplysninger om metoden, der er påpeget i forbindelse med præsenteres resultaterne.

<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 dobbelt-strenget pauser (DSB) er alvorlige DNA læsioner. Analyse af dannelse og reparation af DSB er relevante i et bredt spektrum af forskningsområder herunder genom integritet, genotoksicitet, stråling biologi, aldring, kræft og udvikling af lægemidler. Svar på DSB, er Histon H2AX fosforyleret på Serin 139 i en region af flere megabase par danner diskrete nukleare foci påvises ved immunfluorescens mikroskopi. Desuden er 53BP1 (p53 bindende protein 1) er en anden vigtig DSB-responderende protein fremmer reparation af DSB ved nonhomologous afslutning-tiltræder samtidig forhindre homologe rekombination. Ifølge de specifikke funktioner i γH2AX og 53BP1, kan den kombinerede analyse af γH2AX og 53BP1 ved immunfluorescens mikroskopi være en rimelig tilgang for en detaljeret analyse af DSB. Dette håndskrift giver en trinvis protokol suppleret med metodisk noter til at udføre teknikken. Konkret er indflydelse af cellecyklus på γH2AX foci mønstre demonstreret i normal fibroblaster af cellelinie NHDF. Yderligere, værdien af γH2AX foci som en biomarkør er afbildet i x-ray bestrålet lymfocytter af en sund person. Endelig er genetiske ustabilitet i CD34 + celler af en patient med akut myeloid leukæmi undersøgt ved immunfluorescens mikroskopi af γH2AX og 53BP1.

Analyse af γH2AX foci i celler er mest nøjagtige i den G0/G1 og G2 fase når γH2AX foci vises som adskilte fluorescerende prikker (figur 5A). Derimod kompliceres analyse af γH2AX foci i celler i S-fasen af spredte pan-nukleare γH2AX pletter forårsaget af replikering proces (figur 5B).
Fiksering af celler blev udført med 4% PFA (10 min) og permeabilization med 0,1...

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

Håndskriftet indeholder en protokol til analyse af DNA dobbelt-strenget pauser ved immunfluorescens mikroskopi af γH2AX og 53BP1.

Immunfluorescens mikroskopi af γH2AX og 53BP1 til at analysere den dannelse og reparation af DNA dobbelt-strenget pauser

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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