Il sostegno della Australian Institute of Nuclear Science and Engineering è riconosciuto. TCK è stato il destinatario di premi AINSE. Lab Medicina epigenomic è supportata dal National Health and Medical Research Council of Australia (566.559). LM è supportato da Melbourne di ricerca (Università di Melbourne) e borse di studio integrative Biomedical Imaging CRC. Il supporto di Monash Micro Imaging (Dr. Stephen Cody e Iska Carmichael) è stata preziosa per questo lavoro.

<html> Preparazione delle cellule <ol><li> Cheratinociti umani (FEP-1811) sono state coltivate in cheratinociti-siero Media gratuito (K-SFM; Invitrogen) integrato con il fattore di crescita epidermico, estratto di ipofisi bovina e 20 mg / ml di gentamicina, a 37 ° C e 5% di CO 2. </li><li> Una sospensione singola cella è stata preparata da staccare con tripsina-EDTA (0,05% v / v) </li><li> Le cellule sono state seminate a 8-Tek ben Lab II diapositive microchamber (10.000 cellule / pozzetto) e le diapositive sono state incubate per 3 giorni a 37 ° C e 5% di CO 2. </li></ol> Irradiazione <ol><li> Le cellule sono state irradiate su ghiaccio con 2 Gy utilizzando una sorgente di 137 Cs (Gammacell 1000 Elite irradiatore; Nordion internazionale, ON, Canada, 20,6 secondi / Gy) </li><li> Controllo irradiato e 2 Gy cellule irradiate sono state incubate per 1 ora a 37 ° C e 5% di CO 2. </li></ol> Immunofluorescenza <ol><li> Media è stata una soffiata e le cellule sono state lavate con 300μl di PBS (w / o Ca 2 + o Mg 2 +) per bene e sono stati ruotati su un mixer orbitale per 5 minuti. </li><li> Il buffer è stata una soffiata e 100μl di preparati al momento 4% (v / v) è stato aggiunto paraformaldeide in ciascun pozzetto e diapositive sono state incubate a temperatura ambiente per 10 minuti. Tutte le incubazioni sono state eseguite in una vasca colorazione umidificata </li><li> Le cellule sono state poi lavate con PBS (w / o Ca 2 + o Mg 2 +). I vetrini sono stati posti in una vaschetta Coplin e ruotata su un mixer orbitale per 5 minuti. Questa fase di lavaggio è stata ripetuta altre due volte. </li><li> Il buffer è stata una soffiata e l&#39;eccesso di PBS è stato gentilmente cancellato. </li><li> Le cellule sono state permeabilizzate con 100ml Triton X-100 (0,1% v / v) per bene e una incubazione di 10 minuti a temperatura ambiente. </li><li> Le cellule sono state lavate con PBS (w / o Ca 2 + o Mg 2 +), come descritto nei punti 3 e 4 di cui sopra. </li><li> Non specifico legame con le proteine ​​è stato bloccato con 100 ml di BSA (1% v / v) per bene e 20 minuti di incubazione a temperatura ambiente. </li><li> L&#39;eccesso di BSA è stata una soffiata e 100μl di mouse primario monoclonale anti-fosfo-istone H2AX anticorpi (diluito 1:500 in 1% BSA, Millipore), è stato aggiunto in ciascun pozzetto di una incubazione 1 ora a temperatura ambiente. </li><li> Le cellule sono state lavate con PBS (w / o Ca 2 + o Mg 2 +), come descritto nei punti 3 e 4 di cui sopra e incubato con 100μl di anticorpo secondario (Alexa Fluor 488 di capra anti-IgG di topo diluito 1:500 in BSA 1% ; Invitrogen) per bene per 45 minuti a temperatura ambiente al buio. (Anticorpo diluito è stato tenuto all&#39;oscuro durante tutta la procedura) </li><li> Le cellule sono state lavate con PBS (w / o Ca 2 + o Mg 2 +), come descritto nei punti 3 e 4 di cui sopra. Tuttavia, l&#39;esposizione alla luce è stato minimizzato usando pellicola. </li><li> Di contrasto nucleare è stata eseguita con 100ml TOPRO3 (diluito 1:500; Invitrogen) per bene e una incubazione 10 minuti a temperatura ambiente </li><li> Le cellule sono state lavate con PBS (w / o Ca 2 + o Mg 2 +) come descritto al punto 10. </li><li> Le camere sono state accuratamente rimosse dalle guide di scorrimento, l&#39;umidità in eccesso è stato cancellato e le diapositive sono stati lasciati asciugare all&#39;aria. </li><li> Una goccia di prolungare ORO anti-sbiadimento soluzione (Invitrogen) è stato aggiunto per bene e le diapositive sono state montate (22x50 millimetri coprioggetto) e il liquido in eccesso lungo i bordi di scorrimento è stata cancellata. </li><li> I vetrini sono stati tenuti al buio per altri 30 minuti a temperatura ambiente prima di sigillare con smalto. </li><li> I vetrini sono stati conservati durante la notte a 4 ° C al buio prima dell&#39;analisi. </li></ol> Microscopia / Analisi <ol><li> Meta confocale Zeiss LSM510 Microscpe utilizzato per acquisire immagini utilizzando la GFP standard (per γH2AX - Alexa Fluor 488 capra anti-topo IgG) e laser rosso lontano (per TOPRO-3). Tipicamente, un olio 63 x lente ad immersione obiettivo è utilizzato. Le immagini sono acquisite in una serie Z tracciato con dimensione del passo di 0,5 micron. Un passo di 0,5 micron è stata scelta per minimizzare la perdita di focolai presenti nei diversi piani nei nuclei. Durante l&#39;analisi, i piani individuali sono deconvoluted e sovrapposti per produrre una immagine proiettata al massimo per ridurre al minimo la sovrapposizione di focolai (Top-hat filtro applicato). </li><li> Metamorph (Molecular Devices, USA) è stato utilizzato per analizzare il numero di focolai. Il programma quantitates il numero di focolai in ogni cella dopo la soglia è stata applicata per escludere sfondo. Le informazioni vengono registrate in un foglio di calcolo Microsoft Excel per ulteriori analisi. </li></ol><img src="/files/ftp_upload/1957/1957fig1.jpg" alt="Figura 1" /> Figura 1. Visualizzazione di immunofluorescenza γH2AX focolai (verde) non trattati in cheratinociti umani e in cellule irradiate con 2 Gy e incubate per circa 1 ora a 37 ° C, 5% di CO 2. Il DNA è stato macchiato con TOPRO-3 (blu). Immagini sono state acquisite utilizzando un Zeiss LSM 510 Meta microscopio confocale. Bar = 10 micron. e_content &quot;&gt; <img src="/files/ftp_upload/1957/1957fig2.jpg" alt="Figura 2" /> Figura 2. Visualizzazione di immunofluorescenza γH2AX focolai (verde) in cheratinociti umani e in cellule irradiate con 2 Gy e incubate per circa 1 ora a 37 ° C, 5% di CO 2. Immagini sono state acquisite utilizzando un Zeiss LSM 510 Meta microscopio confocale utilizzando 0,5 micron Z-sezionamento (1-9) per garantire che tutti focolai sono stati acquisiti. Le immagini è stata poi impilati per la quantificazione utilizzando Metamorph. Il DNA è stato macchiato con TOPRO-3 (blu). Il γH2AX impilati e le immagini blu erano accatastati per la visualizzazione. Bar = 10 micron.

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

Nessun conflitto di interessi dichiarati.

In seguito all&#39;esposizione a radiazioni ionizzanti (raggi γ), γH2AX forma foci rapidamente e numeri focolai raggiungono un massimo tra 30-60 minuti 2. Pertanto, il nostro 1 ora dalla irradiazione punto di tempo riflette la formazione iniziale DSB. Abbiamo usato la dose di radiazioni clinicamente rilevante di 2 Gy per il nostro esperimento. Tuttavia, il metodo può essere utilizzato per dosi di radiazioni fino a 4 Gy per il rilevamento di formazione iniziale DSB; sovrapposizione significativa di focolai ...

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

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

DNA a doppio filamento (DSB), che sono indotti da processi metabolici sia endogena o da fonti esogene, sono una delle lesioni del DNA più critici in termini di sopravvivenza e la conservazione dell&#39;integrità del genoma. Una risposta presto per l&#39;induzione di DSB è la fosforilazione della variante dell&#39;istone H2A, H2AX, alla serina-139 residui, altamente conservati nel C-terminale motivo SQEY, formando γH2AX<sup&gt; 1</sup&gt;. Dopo l&#39;induzione di DSB, H2AX è rapidamente fosforilata dalla fosfatidil-inosito 3-chinasi (Pikk) famiglia di proteine, atassia telangiectasia mutati (ATM), DNA-proteina chinasi subunità catalitiche e ATM e RAD3-correlate (ATR)<sup&gt; 2</sup&gt;. Tipicamente, solo poche paia di basi (bp) sono implicati in un DSB, tuttavia, vi è amplificazione del segnale significativo, data l&#39;importanza delle modificazioni della cromatina nella segnalazione del danno al DNA e riparazione. Fosforilazione di H2AX mediato principalmente da ATM si diffonde in aree adiacenti della cromatina, che colpisce circa lo 0,03% del totale dei cellulari H2AX per DSB<sup&gt; 2,3</sup&gt;. Questo corrisponde alla fosforilazione di circa 2000 molecole H2AX spanning ~ 2 regioni Mbp di cromatina che circonda il sito della DSB e provoca la formazione di discreto γH2AX focolai che possono essere facilmente visualizzati e quantificato mediante microscopia di immunofluorescenza<sup&gt; 2</sup&gt;. La perdita di γH2AX a DSB riflette la riparazione, tuttavia, vi è una certa polemica su ciò che definisce la riparazione completa dei DSB, è stato proposto che la ricongiunzione dei due filamenti di DNA è sufficiente però, è stato anche suggerito che la reintegrazione del stato originale di compattazione della cromatina è necessario<sup&gt; 4-8</sup&gt;. La scomparsa di γH2AX coinvolge almeno in parte, defosforilazione da fosfatasi, fosfatasi 2A e fosfatasi 4C<sup&gt; 5,6</sup&gt;. Inoltre, la rimozione di γH2AX di redistribuzione che coinvolgono lo scambio degli istoni con H2A.Z è stata implicata<sup&gt; 7,8</sup&gt;. È importante sottolineare che l&#39;analisi quantitativa della γH2AX foci ha portato ad una vasta gamma di applicazioni nella ricerca medica e nucleare. Qui, dimostriamo il metodo più comunemente utilizzato immunofluorescenza per la valutazione del danno al DNA iniziale di rilevazione e quantificazione di γH2AX focolai nei cheratinociti aderente γ-irradiati umano<sup&gt; 9</sup&gt;.

Watch this Scientific Journal Video about Quantification of Î³H2AX Foci in Response to Ionising Radiation at JoVE.com

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

La quantificazione di DNA a doppio filamento striature formazione γH2AX utilizzando come marcatore molecolare è diventata uno strumento prezioso nel campo della biologia delle radiazioni. Qui mostriamo l&#39;utilizzo di un test di immunofluorescenza per la quantificazione della γH2AX focolai dopo l&#39;esposizione delle cellule alle radiazioni.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

20.3K Views.  The Alfred Medical Research and Education Precinct. La quantificazione di DNA a doppio filamento striature formazione γH2AX utilizzando come marcatore molecolare è diventata uno strumento prezioso nel campo della biologia delle radiazioni. Qui mostriamo l'utilizzo di un test di immunofluorescenza per la quantificazione della γH2AX focolai dopo l'esposizione delle cellule alle radiazioni.

Video: Quantification of γH2AX Foci in Response to Ionising Radiation

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

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) ...

Ricerca

Biologia

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 ...

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

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

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

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

Traslocazione polarizzato di proteine ​​fluorescenti in Xenopus</emEctoderma> in risposta alla segnalazione Wnt

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

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

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

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

Due-e tridimensionale Live Cell Imaging di proteine ​​danno al DNA di risposta

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

Quantification of Orofacial Phenotypes in Xenopus

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

Quantification of Orofacial Phenotypes in Xenopus

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

Quantificazione di orofacciale fenotipi in<em&gt; Xenopus</em

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

Measuring Glutathione-induced Feeding Response in Hydra

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

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

Misurazione glutatione indotta risposta alimentazione a Hydra

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

Quantitation of Endothelial Cell Adhesiveness In Vitro

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

Quantitation of Endothelial Cell Adhesiveness In Vitro

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

La quantificazione di adesività delle cellule endoteliali<em&gt; In Vitro</em

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

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

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

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

Approcci sperimentale per lo studio mitocondriale localizzazione e funzione di un nucleare di cellule ciclo chinasi, Cdk1

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

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

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

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

Esame di proteine ​​legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

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

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

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

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

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

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

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

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

Riepilogo

Esplora Altri Video

Capitoli in questo video

ΓH2AX quantificazione dei Foci, a campioni di tessuto

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

Traslocazione polarizzato di proteine fluorescenti in Xenopus in risposta alla segnalazione Wnt

Due-e tridimensionale Live Cell Imaging di proteine danno al DNA di risposta

Quantificazione di orofacciale fenotipi in

Misurazione glutatione indotta risposta alimentazione a Hydra

La quantificazione di adesività delle cellule endoteliali

Approcci sperimentale per lo studio mitocondriale localizzazione e funzione di un nucleare di cellule ciclo chinasi, Cdk1

Esame di proteine legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in

Quantificazione dei γH2AX Foci in risposta alle radiazioni ionizzanti

Riepilogo

Esplora Altri Video

Capitoli in questo video

ΓH2AX quantificazione dei Foci, a campioni di tessuto

Valutazione della distribuzione spaziale delle γH2AX seguenti radiazioni ionizzanti

Traslocazione polarizzato di proteine ​​fluorescenti in Xenopus in risposta alla segnalazione Wnt

Due-e tridimensionale Live Cell Imaging di proteine ​​danno al DNA di risposta

Quantificazione di orofacciale fenotipi in

Misurazione glutatione indotta risposta alimentazione a Hydra

La quantificazione di adesività delle cellule endoteliali

Approcci sperimentale per lo studio mitocondriale localizzazione e funzione di un nucleare di cellule ciclo chinasi, Cdk1

Esame di proteine ​​legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in

Traslocazione polarizzato di proteine fluorescenti in Xenopus in risposta alla segnalazione Wnt

Due-e tridimensionale Live Cell Imaging di proteine danno al DNA di risposta

Esame di proteine legate al DNA Nascente in cellule di mammifero utilizzando la tecnica BrdU-chip-Slot-occidentale