Name: immunofluorescence microscopy of γh2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks
Uploaded: 2017-11-03T14:00:00.000+00:00
Duration: 10 min 47 s
Description: Watch this Scientific Journal Video about Immunofluorescence Microscopy of Î³H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks at JoVE.com

Il progetto è stato sostenuto dalla Fondazione tedesca José Carreras leucemia (14 DJCLS R/2017).

tutti i metodi descritti qui sono stati approvati dal Comitato etica II della Mannheim facoltà medica dell'Università di Heidelberg. Scritto il consenso informato è stato ottenuto da tutti gli individui.
 1. preparazione dei materiali <ol><li>di soluzione anticoagulante: preparare una soluzione di riserva dell'anticoagulante dell'eparina a 200 UI / mL in sodio cloruro 0,9%. Riempire ciascuna delle provette di raccolta (disegnare il volume 9 mL) con 2 mL di soluzione di riserva dell'anticoagulante prima del prelievo dei campioni di sangue o midollo osseo.</li>
	<li>Soluzione di lisi di cellule rosse: preparare il 10 x soluzione di lisi per cellule rosse con 82,91 g di cloruro di ammonio, bicarbonato di ammonio 7,91 g e 2 mL di soluzione di acido etilendiamminotetracetico 0,5 M ad un pH di 8,0 sciogliendo gli agenti in acqua bidistillata per un volume totale di 1 L.</li>
	<li>Soluzione di fissazione: 8.3 aggiungere µ l di una soluzione di idrossido di potassio di 1 M di paraformaldeide 360 mg (PFA) in un tubo di microtitolo. Riempire il tubo con tamponato fosfato salino (PBS) fino alla tacca 1 mL del tubo e di calore del tubo in un blocco di riscaldamento a 95 ° C per ottenere 1 mL di una soluzione PFA del 36%. Trasferire la soluzione PFA 36% 1 mL in una provetta con una capacità di 15 mL. Aggiungere 8 mL di PBS alla soluzione 1 mL 36% PFA, per ottenere 9 mL di una soluzione madre di PFA 4%. Aliquota del 4% soluzione di riserva di PFA e conservare a-18 ° C fino a utilizzare per la fissazione delle cellule. 
	Attenzione: la soluzione di idrossido di potassio 1) è corrosivo. Essere consapevoli di protezione degli occhi e della pelle. 2) PFA è cancerogeno. Evitare il contatto con la pelle e l'inalazione. Preparare la soluzione di fissaggio sotto un cofano.</li>
	<li>Soluzione di permeabilizzazione: aggiungere 50 µ l octoxinolo 9 a 50 mL di PBS per ottenere una soluzione di circa 0.1% octoxinolo 9.</li>
	<li>Soluzione bloccante: preparare 5% e 2% soluzioni di blocco di miscelazione l'agente bloccante della proteina con PBS e store a 6 &#8211; 8 ° C.</li>
</ol> 2. Preparazione dei campioni <ol><li>fibroblasti in coltura delle cellule: coltivare fibroblasti umani della linea cellulare NHDF in un umidificata 5% CO 2 atmosfera a 37 ° C in RPMI 1640 supplementato con 10% siero fetale di vitello, 4 mM Glutammina e 1% di penicillina/streptomicina.
	<ol><li>Preparazione di un vetrino da microscopio con aderente fibroblasti <ol><li>rimuovere il supporto dal pallone (area di crescita di 75 cm 2) con fibroblasti NHDF in crescita esponenziale. Aggiungere 10 mL di PBS al pallone. Lavare delicatamente i fibroblasti aderenti agitando manualmente la beuta per 1 min.</li>
			<li>Rimuovere il PBS e aggiungere tripsina 1 mL nel pallone. Agitare il matraccio delicatamente per garantire che tutte le cellule aderenti sono coperti dalla tripsina. Rimuovere la tripsina dal pallone. Mettere il pallone in incubatrice per 5 min a 37 ° C.</li>
			<li>Prendere il pallone fuori l'incubatrice e aggiungere 10 mL RPMI 1640 liquido nel pallone. Pipettare il mezzo su e giù più volte per disperdere i fibroblasti.</li>
			<li>Pipetta 0,3 mL dei fibroblasti dispersi su ciascuno dei due campi del vetrino da microscopio e inserire la diapositiva nell'incubatrice per 24 h in modo che i fibroblasti aderiscono alla diapositiva. Per immunostaining di γH2AX e 53BP1 nei nuclei dei fibroblasti, passare alla fase 3.1.</li>
		</ol></li></ol></li> <li>Campioni di pazienti <ol><li>campioni di sangue: utilizzare i tubi di raccolta che sono stati riempiti con 2 mL di soluzione di riserva dell'anticoagulante e aggiungere 7 mL di sangue nelle provette. Conservare i campioni durante la notte a temperatura ambiente (cioè, 18-20 ° C). Il giorno successivo, isolare la fase G0/G1 cellule mononucleari di riposo di centrifugazione in gradiente di densità (punto 2.2.3).</li>
		<li>Campioni del midollo osseo: utilizzare i tubi di raccolta che sono stati riempiti con 2 mL di soluzione di riserva dell'anticoagulante (punto 1.1) e aggiungere midollo osseo 7 mL nelle provette. Conservare i campioni durante la notte a temperatura ambiente (cioè, 18-20 ° C). Il giorno successivo, isolare la fase G0/G1 cellule mononucleari di riposo di centrifugazione in gradiente di densità (punto 2.2.3). Utilizzare microsfere di CD34 e colonne di separazione per l'isolamento delle cellule CD34 + di cell.</li>
		<li>Isolamento delle cellule mononucleari di centrifugazione in gradiente di densità 
		Nota: Cellule mononucleari sono isolate da campioni di sangue e midollo osseo di centrifugazione su gradiente di densità 17 , 18 , 19.
		<ol><li>Diluire il campione di sangue eparinizzato in un rapporto di 1:1 con PBS e il campione di midollo osseo eparinizzata in un rapporto di 1:3 con PBS. Sospendere le cellule delicatamente trascinandoli dentro e fuori la pipetta due volte.</li>
			<li>Aggiungere un volume di mezzo di gradienti di densità in una nuova provetta. Delicatamente strato il sangue diluito o midollo osseo sopra il mezzo del gradiente di densità. Fare attenzione a non per mescolare i due strati.</li>
			<li>Centrifugare la provetta per 30 min a temperatura ambiente e 400 g. x lo strato superiore del plasma prelevare con la pipetta. Poi accuratamente raccolto le cellule mononucleari nello strato di sopra il mezzo del gradiente di densità. Le cellule mononucleari di trasferimento in una nuova provetta.</li>
			<li>Aggiungere almeno tre volumi di PBS a cellule mononucleari nel tubo. Sospendere le cellule delicatamente trascinandoli dentro e fuori la pipetta due volte. Centrifugare la provetta per 10 min a temperatura ambiente e 400 x g.</li>
			<li>Dopo la rotazione, rimuovere il supernatante e aggiungere 10 mL di 4 ° C, 1 x soluzione di lisi di cellule rosse. Risospendere delicatamente le cellule trascinandoli dentro e fuori la pipetta due volte e posizionare il tubo sul ghiaccio per 5 min. Aggiungere 30 mL di PBS a 4 ° C, quindi centrifugare la provetta per 10 min a temperatura ambiente e 400 x g.</li>
		</ol></li> <li>Preparazione dei cytospins <ol><li>preparare due cytospins con cellule mononucleari dei campioni dei pazienti (cioè, un cytospin per γH2AX colorazione di immunofluorescenza e un cytospin per γH2AX e 53BP1 combinato di immunofluorescenza) mediante centrifugazione (300 x g, 10 min) di 1.0 x 10 5 celle per ogni preparazione ( Figura 3 e 4).</li>
		</ol></li></ol></li></ol> 3. γH2AX e colorazione di immunofluorescenza 53BP1 <ol><li>fissazione e permeabilizzazione: fissare le cellule con 200 µ l di 4% PFA per 10 min. Dopo la fissazione, lavare delicatamente le cellule tre volte con 30 mL di PBS per 5 minuti ciascuno su un agitatore di laboratorio. Quindi permeabilize le cellule con 200 µ l di 0,1% octoxinolo 9 per 10 min. lavare le cellule delicatamente tre volte con 30 mL di soluzione bloccante 5% per 5 minuti ciascuno su un agitatore di laboratorio. Bloccare le cellule in 30 mL di fresco 5% soluzione per 1 h. di blocco</li>
	<li>Incubazione con gli anticorpi <ol><li>Incubare una preparazione delle cellule con un anticorpo monoclonale anti-γH2AX di topo (1: 500) e l'altra preparazione delle cellule con un anticorpo monoclonale anti-γH2AX di topo (1: 500) e una anticorpo policlonale del coniglio di anti-53BP1 (1: 500) durante la notte a 4 ° C.</li>
		<li>Dopo incubazione lavare le cellule delicatamente 3 volte con 30 mL di soluzione bloccante 2% per ogni 5 minuti su un agitatore di lab.</li>
		<li>Rimuovere la soluzione di blocco e incubare la prima preparazione delle cellule con un anticorpo secondario anti-topo di capra Alexa488-coniugati (1: 500) e la seconda preparazione delle cellule con un (anticorpo secondario anti-topo di capra Alexa488-coniugato 1: 500) e un asino Alexa555-coniugato anticorpo secondario anti-coniglio (1: 500) per 1 h a temperatura ambiente.</li>
	</ol></li> <li>Mezzo di montaggio: mettere il vetrino con le cellule in una ciotola e lavare le cellule delicatamente tre volte con 30 mL di PBS per ogni 5 minuti su un agitatore di laboratorio. Rimuovere il PBS e montare le cellule con mezzo di montaggio contenente 4,6-diamidino-2-phenylindole. Prudentemente messo un vetrino coprioggetto sopra il mezzo di montaggio in modo che bolle d'aria non sono incorporato. Attendere almeno 3 ore per l'indurimento di mezzo di montaggio prima di analizzare le cellule mediante microscopia a fluorescenza.</li>
</ol> 4. Analisi di γH2AX e 53BP1 i fuochi <ol><li>microscopia di fluorescenza: analizzare i fuochi γH2AX e 53BP1 nei nuclei delle cellule con un microscopio a fluorescenza dotato di filtri per DAPI, Alexa 488 e Cy3 durante la formazione immagine a un obiettivo 100x ingrandimento. Registrare immagini con una macchina fotografica ed elaborare le immagini con software di imaging appropriato.</li>
</ol>

<ol>
	<li>Hoeijmakers, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+maintenance+mechanisms+for+preventing+cancer.">Genome maintenance mechanisms for preventing cancer.</a> Nature. 411 (6835), 366-374 (2001).</li><li>Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+and+decay+of+the+primary+structure+of+DNA.">Instability and decay of the primary structure of DNA.</a> Nature. 362 (6422), 709-715 (1993).</li><li>Zeman, M. K., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+replication+stress.">Causes and consequences of replication stress.</a> Nat Cell Biol. 16 (1), 2-9 (2014).</li><li>. Biological effects of low doses of ionizing radiation: A fuller picture Available from: <a target="_blank" href="https://www.iaea.org/sites/default/files/36405843745.pdf">https://www.iaea.org/sites/default/files/36405843745.pdf</a> (1994)</li><li>Vilenchik, M. M., Knudson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+DNA+double-strand+breaks:+production,+fidelity+of+repair,+and+induction+of+cancer.">Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer.</a> Proc Natl Acad Sci U S A. 100 (22), 12871-12876 (2003).</li><li>Simsek, D., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+end-joining+is+suppressed+by+the+canonical+NHEJ+component+Xrcc4-ligase+IV+during+chromosomal+translocation+formation.">Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation.</a> Nat Struct Mol Biol. 17 (4), 410-416 (2010).</li><li>Weinstock, D. M., Brunet, E., Jasin, M. <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+NHEJ-derived+reciprocal+chromosomal+translocations+does+not+require+Ku70.">Formation of NHEJ-derived reciprocal chromosomal translocations does not require Ku70.</a> Nat Cell Biol. 9 (8), 978-981 (2007).</li><li>Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., 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=DNA+double-stranded+breaks+induce+histone+H2AX+phosphorylation+on+serine+139.">DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.</a> J Biol Chem. 273 (10), 5858-5868 (1998).</li><li>Scully, R., Xie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+strand+break+repair+functions+of+histone+H2AX.">Double strand break repair functions of histone H2AX.</a> Mutat Res. 750 (1-2), 5-14 (2013).</li><li>Walters, D. 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. 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=Increase+of+DNA+damage+and+alteration+of+the+DNA+damage+response+in+myelodysplastic+syndromes+and+acute+myeloid+leukemias.">Increase of DNA damage and alteration of the DNA damage response in myelodysplastic syndromes and acute myeloid leukemias.</a> Leuk Res. 57, 112-118 (2017).</li><li>Panier, S., Boulton, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+break+repair:+53BP1+comes+into+focus.">Double-strand break repair: 53BP1 comes into focus.</a> Nat Rev Mol Cell Biol. 15 (1), 7-18 (2014).</li><li>Escribano-Diaz, C., 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=A+cell+cycle-dependent+regulatory+circuit+composed+of+53BP1-RIF1+and+BRCA1-CtIP+controls+DNA+repair+pathway+choice.">A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.</a> Mol Cell. 49 (5), 872-883 (2013).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+White+Blood+Cells.">Separation of White Blood Cells.</a> Nature. 204, 793-794 (1964).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+and+granulocytes+from+human+blood.+Isolation+of+monuclear+cells+by+one+centrifugation,+and+of+granulocytes+by+combining+centrifugation+and+sedimentation+at+1+g.">Isolation of mononuclear cells and granulocytes from human blood. 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. 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>

Gli autori non hanno nulla a rivelare.

Microscopia di immunofluorescenza di γH2AX e 53BP1 è un metodo utile per analizzare la formazione e la riparazione di DSB in un ampio spettro di settori di ricerca. I parametri critici che influenzano il risultato degli esperimenti sono la fase del ciclo cellulare, gli agenti utilizzati per la fissazione e la permeabilizzazione delle cellule, la scelta di anticorpi e l'hardware e il software di microscopio di fluorescenza.
L'influenza del ciclo cellulare sui modelli i fuochi di γH2AX è sta...

DNA è continuamente danneggiato da endogeno (ad es., stress replica, specie reattive dell'ossigeno, intrinseca instabilità del DNA) ed esogene (ad esempio, radicali chimici, irradiazione) fonti (Figura 1)1,2 ,3,4. Tra il danno del DNA, rotture del DNA a doppio filamento (DSB) sono lesioni particolarmente gravi e possono indurre la morte delle cellule o carcinogenesi. Circa 50 DSB possono sorgere al cellulare e ciclo cellulare5. In cellule di mammifero, ricombinazione omologa (HR) e fine-unirsi nonhomologous (NHEJ) sviluppato come vie principali per la riparazione di DSB (Figura 2). HR si verifica nella fase tarda S/G2 e utilizza un'intatta sorella cromatidio come modello per la riparazione potenzialmente privo di errori. In confronto, NHEJ è attivo durante tutto il ciclo cellulare e potenzialmente mutageni come coppie di basi può essere aggiunto o resecate prima legatura dell'estremità rotte. Inoltre, fine-entrare in alternativa possono essere assunti come un meccanismo di lento mutagene backup ripristino in caso di carenza NHEJ6,7.
DSB inducono la fosforilazione dell'istone H2AX a serina 139 in una regione di diverse coppie di megabase intorno ogni DSB. I fuochi che formanti nucleari sono denominati γH2AX i fuochi e rilevabili da microscopia di immunofluorescenza8. ΓH2AX promuove l'assunzione di ulteriori proteine DSB-sensible a reagire ed è coinvolto nel rimodellamento della cromatina, riparazione del DNA e di trasduzione di segnale9. Come ogni focus γH2AX è considerato per rappresentare un singolo DSB, la quantificazione del DSB da microscopia di immunofluorescenza è possibile, che è stata dimostrata in linee cellulari del cancro e i campioni dei pazienti10,11, 12 , 13 , 14. 53BP1 (proteina p53 1) è un'altra proteina chiave nel mediare la riparazione DSB. È coinvolto nel reclutamento di DSB-sensible a reagire proteine, segnalazione di checkpoint e le sinapsi di DSB estremità15. Inoltre, 53BP1 svolge un ruolo critico nella scelta di percorso di riparazione DSB. Si innesca la riparazione del DSB verso NHEJ mentre HR è impedito16. Considerando le funzioni originali di γH2AX e 53BP1 in riparazione DSB, l'analisi simultanea di γH2AX e 53BP1 da microscopia di immunofluorescenza può essere un metodo utile per l'analisi precisa della formazione e della riparazione del DSB.
Questo manoscritto fornisce un protocollo dettagliato per l'esecuzione di microscopia di immunofluorescenza di γH2AX e 53BP1 nei nuclei delle cellule. In particolare, la tecnica è applicata in fibroblasti normali della linea cellulare NHDF, nei linfociti irradiati raggi x di un individuo sano e in cellule CD34 + di un paziente con la leucemia mieloide acuta. I dettagli del metodo sono indicati nel contesto dei risultati presentati.

<table><tbody><tr><td>RPMI medium</td><td>Sigma-Aldrich</td><td>R0883</td><td>Medium for cell culture</td></tr><tr><td>Heparin sodium</td><td>ratiopharm</td><td>PZN 3029843</td><td>&amp;nbsp;Heparin 5,000 I.U. / mL</td></tr><tr><td>Sodium chloride solution 0.9%</td><td>B. Braun</td><td>PZN 1957154</td><td>Component of the anticoagulant stock solution</td></tr><tr><td>Ficoll-Paque Premium</td><td>GE Healthcare</td><td>17-5442-02</td><td>Medium for isolation of mononuclear cells</td></tr><tr><td>Trypsin solution 10X</td><td>Sigma-Aldrich</td><td>59427C</td><td>Enzyme for dissociation of fibroblasts in cell culture</td></tr><tr><td>CD34 MicroBead Kit</td><td>Miltenyi Biotec</td><td>130-046-702</td><td>Isolation of CD34+ myeloid progenitor cells</td></tr><tr><td>Diagnostic microscope slides</td><td>Thermo Scientific</td><td>ER-203B-CE24</td><td>Microscope slides</td></tr><tr><td>Megafuge 1.0 R</td><td>Heraeus</td><td>75003060</td><td>&amp;nbsp;Tabletop centrifuge&amp;nbsp;</td></tr><tr><td>Cytospin device</td><td></td><td></td><td></td></tr><tr><td>Lid</td><td>Heraeus</td><td>76003422</td><td>Lid for working without micro-tubes</td></tr><tr><td>Cyto container</td><td>Heraeus</td><td>75003416</td><td>Cyto container with 2 conical bores</td></tr><tr><td>Clip carrier</td><td>Heraeus</td><td>75003414</td><td>Carrier for holding a cyto container and a slide</td></tr><tr><td>Support insert</td><td>Heraeus</td><td>75003417</td><td>Support insert for holding a clip carrier</td></tr><tr><td>Triton X-100 (Octoxinol 9)</td><td>Thermo Scientific</td><td>85112</td><td>Detergent for permeabilization of cell membranes</td></tr><tr><td>Potassium hydroxide solution 1M</td><td>Merck Millipore</td><td>109107</td><td>Necessary for preparing the paraformaldehyde solution</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Fixation agent</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>Balanced salt solution</td></tr><tr><td>Chemiblocker</td><td>Merck Millipore</td><td>2170</td><td>Blocking agent</td></tr><tr><td>Mouse monoclonal anti-&amp;gamma;H2AX antibody (JBW301)</td><td>Merck Millipore</td><td>05-636</td><td>Primary antibody for detection of &amp;gamma;H2AX</td></tr><tr><td>Polyclonal rabbit anti-53BP1 antibody (NB100-304)</td><td>Novus Biologicals</td><td>NB100-304</td><td>Primary antibody for detection of 53BP1</td></tr><tr><td>Alexa Fluor 488-conjugated goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11001</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 555-conjugated donkey anti-rabbit antibody</td><td>Invitrogen</td><td>A-31572</td><td>Secondary antibody</td></tr><tr><td>Vectashield mounting medium</td><td>Vector Laboratories</td><td>H-1200</td><td>Contains DAPI for staining of DNA</td></tr><tr><td>Axio Scope.A1</td><td>Zeiss</td><td>490035</td><td>Fluorescence microscope</td></tr><tr><td>Cool Cube 1 CCD camera</td><td>Metasystems</td><td>H-0310-010-MS</td><td>Camera system for digital recording</td></tr><tr><td>Isis software</td><td>Metasystems</td><td>Not applicable</td><td>Microscope software</td></tr></tbody></table>

immunofluorescence microscopy of &#947;h2ax and 53bp1 for analyzing the formation and repair of dna double-strand breaks

Rotture del DNA a doppio filamento (DSB) sono gravi lesioni al DNA. Analisi della formazione e della riparazione del DSB sono rilevante in un ampio spettro di settori di ricerca tra cui integrità del genoma, genotossicità, radiobiologia, invecchiamento, cancro e lo sviluppo di farmaci. In risposta a DSB, istone H2AX è fosforilata a serina 139 in una regione di diverse coppie di megabase formando fuochi nucleari discreti rilevabili da microscopia di immunofluorescenza. In aggiunta, 53BP1 (proteina p53 1) è un'altra importante proteina DSB-sensible a reagire promuovendo la riparazione del DSB unendo nonhomologous fine-evitando la ricombinazione omologa. Secondo le specifiche funzioni di γH2AX e 53BP1, l'analisi combinata di γH2AX e 53BP1 da microscopia di immunofluorescenza può essere un approccio ragionevole per un'analisi dettagliata del DSB. Questo manoscritto fornisce un protocollo dettagliato completato con metodiche note per l'esecuzione della tecnica. In particolare, l'influenza del ciclo cellulare sui modelli di fuochi γH2AX è dimostrata in fibroblasti normali della linea cellulare NHDF. Inoltre, il valore dei fuochi γH2AX come biomarcatore è raffigurato nei linfociti irradiati raggi x di un individuo sano. Infine, l'instabilità genetica è studiata in cellule CD34 + di un paziente con la leucemia mieloide acuta da microscopia di immunofluorescenza di γH2AX e 53BP1.

Analisi dei fuochi di γH2AX in cellule è più accurata in fase G0/G1 e la fase G2 quando i fuochi di γH2AX vengono visualizzati come punti fluorescenti distinti (Figura 5A). Al contrario, analisi dei fuochi di γH2AX nelle cellule durante la fase S sono complicata da macchioline dispersi γH2AX pan-nucleare causate dal processo di replica (figura 5B).
Fissazione dell...

Watch this Scientific Journal Video about Immunofluorescence Microscopy of Î³H2AX 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

Questo manoscritto fornisce un protocollo per l'analisi delle rotture del doppio filamento di DNA da microscopia di immunofluorescenza di γH2AX e 53BP1.

Microscopia di immunofluorescenza di γH2AX e 53BP1 per analizzare la formazione e la riparazione di rotture a doppio filamento del DNA

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

16.1K Views.  Medical Faculty Mannheim of the Heidelberg University. Questo manoscritto fornisce un protocollo per l'analisi delle rotture del doppio filamento di DNA da microscopia di immunofluorescenza di γH2AX e 53BP1.

Video: Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

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.

Che caratterizzano i processi di riparazione del DNA alle rotture del DNA transitori e duraturo doppio filo da microscopia di immunofluorescenza

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Ricerca

Ricerca sul cancro

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.

Immunofluorescenza Immagini di danno al DNA e riparazione Foci nelle cellule tumorali del colon umano

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Un metodo di punteggio microscopico automatizzato per il test foci γ-H2AX nei linfociti del sangue periferico umano

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Doppia immunofluorescenza di γH2AX e 53BP1 nei linfociti periferici umani

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

Genetica

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

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

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

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Biologia

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.

ΓH2AX quantificazione dei Foci, a campioni di tessuto

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

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.

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

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 ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Visualizzazione dell'interazione delle proteine di riparazione del DNA per immunofluorescenza

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

<meta charset="utf8"></head><body>Valutazione dell'interazione e della localizzazione delle proteine nel danno al DNA

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

<meta charset="utf8"></head><body>Autore in primo piano: Combinazione del saggio del ligando di prossimità con la colorazione Gamma-H2AX per caratterizzare le interazioni proteiche nella risposta a

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

Saggio del ligando di prossimità per localizzare le proteine nei siti di danno al DNA

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

Microscopia di immunofluorescenza di γH2AX e 53BP1 per analizzare la formazione e la riparazione di rotture a doppio filamento del DNA

Riepilogo

Esplora Altri Video

Capitoli in questo video

Che caratterizzano i processi di riparazione del DNA alle rotture del DNA transitori e duraturo doppio filo da microscopia di immunofluorescenza

Immunofluorescenza Immagini di danno al DNA e riparazione Foci nelle cellule tumorali del colon umano

Un metodo di punteggio microscopico automatizzato per il test foci γ-H2AX nei linfociti del sangue periferico umano

Doppia immunofluorescenza di γH2AX e 53BP1 nei linfociti periferici umani

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

ΓH2AX quantificazione dei Foci, a campioni di tessuto

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

Visualizzazione dell'interazione delle proteine di riparazione del DNA per immunofluorescenza

Saggio del ligando di prossimità per localizzare le proteine nei siti di danno al DNA

Saggio del ligando di prossimità per localizzare le proteine nei siti di danno al DNA