Ci piace riconoscere Marcelo Toro per l&#39;assistenza con la citometria a flusso. Questo studio è stato sostenuto dalla svedese Heart-Lung Foundation e, Commissione UE FP7 &quot;CardioCell&quot;, Consiglio svedese della ricerca, AFA assicurazioni e ALF. OB è stato sostenuto da Deutsche Forschungsgemeinschaft.

1. Isolamento dei Nuclei Cardiac <ol><li> Coat tubi ultracentrifuga (Tubi centrifuga Beckman # 363664) con 10 ml di 1% BSA / PBS soluzione di rivestimento. Coprire le provette e farli ruotare per 30 minuti in un rotatore tubo. Rimuovere la soluzione di rivestimento e lasciare la centrifuga tubi aria secca (un tubo per il cuore di topo è richiesto per l&#39;analisi di cuore di topo singoli, alternativamente fino a 5 cuore di topo o 1 g di tessuto cardiaco da una specie diversa (ad esempio umano) può essere elaborato in un tubo). </li><li> Tutti i passaggi seguenti devono essere eseguiti sul ghiaccio. Sezionare il ventricolo sinistro del mouse dal cuore fresco o snap-frozen con un bisturi. Attenzione, questo protocollo è ottimizzato per il cuore del mouse, ma può anche essere adattato per ratto o cuore umano. In alternativa, utilizzare fino a 1 g di tessuto cardiaco da una specie diversa. </li><li> Tagliare il campione in cubicoli di piccole dimensioni con un bisturi. </li><li> Trasferire i pezzi di tessuto in un tubo Falcon da 50 ml riempito con 15 ml di tampone di lisi. </li><li> Omogeneizzare latessuto cardiaco con un T-25 Ultra-Turrax sonda omogeneizzatore (IKA) a 24.000 rpm per 10 sec. </li><li> Diluire l&#39;omogeneizzato con un volume uguale di tampone di lisi a 30 ml. </li><li> Utilizzare un bicchiere douncer (40 ml) per omogeneizzare ulteriormente il tessuto e il libero nuclei. Eseguire otto colpi con un pestello gioco di grandi dimensioni. </li><li> Passare il greggio isolare nuclei attraverso un 100 micron e 70 micron nylon mesh cellulare filtro (BD Biosciences), consecutivamente. </li><li> Centrifugare la nuclei grezzo isolato in una centrifuga refrigerata (4 ° C) a 700 xg per 10 min. </li><li> Rimuovere il surnatante attentamente invertendo i tubi e pulire l&#39;interno del tubo con carta assorbente. Fare attenzione a non disturbare il pellet nuclei. </li><li> Sciogliere il nuclei grezzo isolato in 5 ml di tampone saccarosio pipettando la soluzione più volte su e giù. Aggiungi un ulteriore 25 ml di tampone saccarosio alla disciolta pellet. </li><li> Aggiungere 10 ml di tampone saccarosio appena preparato al tubo rivestito ultracentrifuga (vedi punto 1.1). &lt;/ Li&gt; <li> Accuratamente sovrapporre il aggiunti 10 ml di tampone saccarosio con i nuclei pellet risospeso sciolto in tampone saccarosio dal passaggio 1,9. </li><li> Bilanciare le provette prima di metterli in un rotore JS13.1 libera oscillazione e posizionare il rotore in una centrifuga ad alta velocità (Beckman Avanti S-25). </li><li> Spin il campione nuclei a 13.000 xga 4 ° C per 60 min. </li><li> Quando la rotazione è stata completata, rimuovere i tubi accuratamente dal rotore e scartare il supernatante invertendo i tubi e pulire i detriti restante dall&#39;interno dei tubi con carta assorbente. </li><li> Sciogliere i nuclei pellet in 1 ml di tampone nuclei di stoccaggio (NSB più buffer). Nota: NSB più contiene 1,5 mM spermina come stabilizzante DNA. </li><li> Procedere con il punto 2.1, immunocolorazione dei nuclei cardiomiociti. </li></ol> 2. Immunoenzimatica per Citometria a Flusso <ol><li> Preparare il controllo negativo per immunocolorazione. Prelevare un &#39;aliquota di 20 microlitri di fuori del campione nuclei edd 980 pl di tampone NSB più. </li><li> Aggiungi anti-pericentriolar materiale 1 anticorpi (di coniglio anti-PCM-1, anticorpi Atlas) al campione nuclei in una diluizione di 1:500 per nuclei immunolabel cardiomiociti. Aggiungere l&#39;anticorpo isotipo nella stessa diluizione come l&#39;anti-anticorpo PCM-1 per il controllo negativo, preparato nella fase 2,1. </li><li> Incubare controllo negativo e la provetta a 4 ° C per una notte. </li><li> Lavare controllo negativo e campione almeno una volta con NSB più tampone (spin down tubi in una centrifuga refrigerata (4 ° C) a 700 xg per 10 min. Eliminare il surnatante e sciogliere il nuclei pellet in 1 ml di tampone più NSB). </li><li> Aggiungi anti-coniglio immunofluorescenza secondario (FITC o APC) al controllo negativo e la provetta in una diluizione di 1:1000. </li><li> Incubare controllo negativo e la provetta a 4 ° C per 1 h. </li><li> Lavare controllo negativo e campione almeno una volta con NSB più tampone (spin down tubi in una centrifuga refrigerata (4 ° C) a 700 xg per 10 min. Gettare il surnatante e sciogliere i nuclei pellet in 1 ml di NSB più buffer). </li><li> Procedere con l&#39;analisi di citometria di flusso e di smistamento. </li></ol> 3. Citometria a Flusso <ol><li> Coat nuclei provette Falcon (15 ml) con 1% BSA / PBS soluzione prima di iniziare la citometria a flusso di smistamento come descritto al punto 1,1. </li><li> Filtrare il campione e il controllo negativo tramite un setaccio cella 30 um e caricare il primo controllo negativo al citofluorimetro (Influx BD). Definire il primo cancello e la seconda per definire nuclei e canottiere (nuclei singola), sulla base di forward scatter (FSC), larghezza di impulso in avanti scatter (FS larghezza d&#39;impulso) e Side Scatter (SSC) (Fig. 2a e b). Aggiunta di un DNA macchia (DRAQ5 (1:500)) per il campione può aiutare a identificare la popolazione nuclei inizialmente. </li><li> Caricare il campione immunolabeled e definire la terza porta per isolare nuclei cardiomiociti (PCM-1-positve) da non-cardiomiociti nuclei (PCM-1-negativo). Avviare l&#39;ordinamento(Fig. 2c e d). </li></ol> Optional: Al fine di analizzare il contenuto nucleare di DNA (ploidia) e di eseguire l&#39;analisi del ciclo cellulare aggiungere un DNA adeguato macchia per i nuclei (ad esempio, Hoechst 33342 o DRAQ5) (Fig. 2e). <ol start="4"><li> Dopo cernita citometria di flusso, posizionare i nuclei su ghiaccio e ri-analisi per determinare la purezza di smistamento (Fig. 3a e b). </li><li> Centrifugare la nuclei ordinato nei tubi di raccolta a 1500 xg in una centrifuga refrigerata per 15 min. </li><li> Sciogliere il nuclei pellet in un buffer compatibile con l&#39;applicazione a valle. </li></ol> 4. Risultati rappresentativi Nuclei morfologia e l&#39;integrità può essere valutata attraverso macchie di DNA e visualizzati al microscopio (Fig. 1). Successo PCM-1 di etichettatura può essere valutata mediante microscopia in epifluorescenza e mediante citometria a flusso (Fig. 1 e Fig. 2c e d). PCM-1-positiVE e popolazioni negativi devono essere ben separati uno dall&#39;altro (Fig. 2c e d). In murino ventricolo sinistro circa il 30% di tutti i nuclei dovrebbe essere nuclei cardiomiociti (Fig. 2d). Ordinamento purezza può essere valutata da ri-analizzando i nuclei ordinati (Fig. 3a e b). Entrambe le popolazioni nuclei dovrebbe avere una purezza superiore al 95% di smistamento. <img alt="Figura 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Figura 1. PCM-1 identifica nuclei cardiomiociti. Nuclei cardiaca (a) vengono colorate con anticorpi anti PCM-1 (b) e per Nkx2.5 (c) in un cuore topo adulto. (D) PCM-1-marcato nuclei sono circondati da citoplasma cardiomiociti (catena pesante della miosina (MHC)) ed esprimere il fattore di trascrizione Nkx2.5, che documenta l&#39;identificazione accurata dei nuclei cardiomiociti da PCM-1 colorazione (scala di 20 bar e 10 micron micron (d, inserto)). (E) isolati nuclei cardiache visualizzati con il DNA macchia DRAQ5. (F e G) Cardiomyocnuclei yte sono etichettati con anticorpi contro il PCM-1 (scala bar 10 micron). Nota, il pattern di colorazione epinuclear PCM-1 in nuclei muscolari in sezione del tessuto e in nuclei isolati (frecce). <img alt="Figura 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Figura 2. Citometria a flusso ordinamento dei nuclei cardiomiociti. (A) nuclei Cardiac sono identificati dalla forward scatter (FSC) e lato scatter (SSC). (B) Una seconda porta identifica i singoli nuclei da FSC e FS larghezza di impulso 5. (C, d) gating fluorescente permette la separazione dei nuclei cardiomiociti (PCM-1-positive) e non-cardiomiociti (PCM-1-negativi) nuclei di tessuto cardiaco. (E) cardiomiociti mouse sono in gran parte (&gt; 80%) diploide (2n), solo un piccolo sottoinsieme è tetraploide (4n) 6. Nota, cardiomiociti umani contengono una maggiore frequenza di nuclei poliploidia (&gt; 2n) 7,8. <img alt="Figura 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Figura 3. Analisi di purezza ordinati nuclei cardiomiocita e non cardiomiociti. Re-analisi di ordinato nuclei non cardiomiociti (a) e cardiomiociti (b). Entrambe le popolazioni mostrano una purezza superiore al 99% di smistamento.

<ol>
	<li>Ang, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> American journal of physiology. Cell physiology. , 298-1603 (2010).</li><li>Bergmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardiomyocyte+nuclei+and+assessment+of+ploidy+for+the+analysis+of+cell+turnover.">Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.</a> Experimental cell research. 327, 188-194 (2011).</li><li>Bergmann, O. <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+Cardiomyocyte+Renewal+in+Humans.">Evidence for Cardiomyocyte Renewal in Humans.</a> Science. 324, 98-102 (1126).</li><li>Walsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+cell+cycle+control+and+growth+estimation.">Cardiomyocyte cell cycle control and growth estimation.</a> Cardiovascular Research. , 1-31 (2010).</li><li>Spalding, K., Bhardwaj, R. D., Buchholz, B., Druid, H., Frisén, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Adler, C. P., Friedburg, H., Herget, G. W., Neuburger, M., Schwalb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+cardiomyocyte+DNA+content,+ploidy+level+and+nuclear+number+in+mammalian+hearts.">Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts.</a> Virchows Arch. 429, 159-164 (1996).</li><li>Herget, G. W., Neuburger, M., Plagwitz, R., Adler, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRPA+is+a+specific+cell-surface+marker+for+isolating+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.</a> Nature. 29, 1011-1018 (2011).</li><li>Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes.">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature Methods. 7, 61-66 (2010).</li><li>Fransioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+c-kit-Positive+Cell+Response+to+Pathological+Challenge+in+the+Myocardium.">Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium.</a> Stem Cells. 26, 1315-1324 (2008).</li><li>Elliott, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKX2-5(eGFP/w)+hESCs+for+isolation+of+human+cardiac+progenitors+and+cardiomyocytes.">NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.</a> Nature Methods. 8, 1037-1040 (2011).</li><li>Laflamme, M. A. <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+Cardiomyocyte+Repopulation+by+Extracardiac+Progenitors+in+Transplanted+Human+Hearts.">Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts.</a> Circulation Research. 90, 634-640 (2002).</li><li>Srsen, V., Fant, X., Heald, R., Rabouille, C., Merdes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+proteins+form+an+insoluble+perinuclear+matrix+during+muscle+cell+differentiation.">Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.</a> BMC cell biology. 10, 28 (2009).</li><li>Spoelgen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometry-based+technique+to+measure+adult+neurogenesis+in+the+brain.">A novel flow cytometry-based technique to measure adult neurogenesis in the brain.</a> Journal of neurochemistry. 119, 165-175 (2011).</li><li>Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+DNA+synthesis+and+binucleation+during+murine+development.">Cardiomyocyte DNA synthesis and binucleation during murine development.</a> The American journal of physiology. 271, H2183-H2189 (1996).</li><li>Olivetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+cardiac+hypertrophy+and+ischemic+cardiomyopathy+do+not+affect+the+proportion+of+mononucleated+and+multinucleated+myocytes+in+the+human+heart.">Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart.</a> J Mol Cell Cardiol. 28, 1463-1477 (1996).</li><li>Okada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+sorting+of+neuronal+and+glial+nuclei+from+central+nervous+system+tissue.">Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue.</a> Journal of cellular physiology. 226, 552-558 (2011).</li><li>Matevossian, A., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Nuclei+Isolation+from+Human+Postmortem+Brain+Tissue.">Neuronal Nuclei Isolation from Human Postmortem Brain Tissue.</a> J. Vis. Exp. (20), e914 (2008).</li></ol>

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Identificazione accurata di nuclei cardiomiocita è cruciale per l&#39;analisi dei processi rigenerativi nel miocardio 2,3. Le tecniche convenzionali per isolare cardiomiociti di tessuto fresco si basa principalmente sulla digestione enzimatica delle proteine ​​della matrice extracellulare e la successiva purificazione dalle cellule interstiziali mediante centrifugazione a bassa velocità. Ulteriore purificazione di cardiomiociti viventi di cellule staminali embrionali (ESC) può essere eseguita da immunomarcatura con marcatori superficiali quali SIRPA 9 o coloranti mitocondriali 10, cardiomiociti fissi possono essere identificati dai marcatori citoplasmatici quali catena pesante della miosina (MHC) o proteine ​​nucleari come GATA4 o Nkx2.5. Tuttavia, l&#39;espressione di Nkx2.5 GATA4 e non è limitato a cardiomiociti maturi, ma può essere anche rilevata nelle cellule progenitrici durante lo sviluppo e dopo infarto cardiaco 11,12. Strategie genetiche per identificare cardiomiociti mostrare la più alta specificità, ma cAnnot essere applicato in modelli animali o esseri umani grandi 1. I limiti delle strategie esistenti hanno portato a conclusioni controverse basandosi sulla quantificazione di eventi rari, quali la proliferazione, l&#39;apoptosi e chimerismo 1,13. Quindi, vi è la necessità di stabilire una strategia accurata per identificare e purificare maturo nuclei differenziata dei cardiomiociti. Qui, si descrive una tecnica di purificazione basato su materiale pericentriolar 1 (PCM-1). In precedenza, abbiamo rafforzato la validità del nostro isolamento nuclei mostrando che tre marcatori indipendenti nucleari, PCM-1, la troponina cardiaca T e I, identificare la popolazione stessa dei cardiomiociti. 2,3 La popolazione purificato ha mostrato un arricchimento elevato di cardiomiociti prodotti genici specifici come come troponine cardiache, proteine ​​della catena pesante della miosina e il fattore di trascrizione Nkx2.5 e GATA4 2,3. Il protocollo attuale, basato sui PCM-1, è stato applicato con successo fo l&#39;analisi di umano e di topo turnover cardiomyocyte 2,4 nonché per l&#39;analisi del profilo cardiomiociti trascrizionale 2. Anticipiamo che la strategia presentata può anche essere adattato all&#39;isolamento dei cardiomiociti da altre specie. PCM-1, una proteina centrosoma, ri-localizza la membrana nucleare cui si accumula e forma una matrice insolubile solo in miociti differenziati 2,14 (Fig. 1). Citometria di flusso consente l&#39;analisi rapida corretto di un elevato numero di cellule, che è richiesto per quantificare eventi rari in cardiomiociti come apoptosi e ciclo cellulare rientro 15. Inoltre, analoghi nucleotidici (ad es BrdU) saggi di proliferazione base può essere facilmente applicato utilizzando la nostra tecnica presentato 4. La tecnica ha il vantaggio che il contenuto di DNA nuclei cardiomiociti singolo (nucleare ploidia) possono essere analizzati in cardiomiociti multinucleari 16,17, che fornisce informazioni importantidi discriminare tra cardiomiociti duplicazione e poliploidizzazione 6-8. Tuttavia, a causa della natura della nostra strategia, una caratterizzazione di multinucleazione cardiomiocita non è possibile. Ulteriori applicazioni del metodo presentato sono studi focalizzati sui cambiamenti epigenetici della cromatina, nucleoproteine, interazioni protein-DNA/RNA e sul nucleare trascrittoma 18,19. A causa della stabilità di nuclei in congelare scongelati-tessuto, la strategia di isolamento presentato nuclei possono essere applicati anche ai tessuti congelati, rendendo possibile utilizzare materiale archiviato da biobanche.

<table border="1"><tbody><tr><td> 1. Lysis Buffer </td></tr><tr><td> Nome del reattivo </td></tr><tr><td> 0,32 M di saccarosio </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl 2 </td></tr><tr><td> 5 mM acetato di magnesio </td></tr><tr><td> 2,0 mM EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Saccarosio tampone </td></tr><tr><td> Nome del reattivo </td></tr><tr><td> 2,1 M saccarosio </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM acetato di magnesio </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Nuclei soluzione per la conservazione (NSB plus) </td></tr><tr&gt; <td> Nome del reattivo </td></tr><tr><td> 0,44 M di saccarosio </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM spermina </td></tr></tbody></table><table border="1"><tbody><tr><td> Reagenti e materiale </td><td> Azienda </td></tr><tr><td> Isotipo IgG di coniglio ChIP-Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Coniglio anti-PCM-1 anticorpo, # HPA023374 </td><td> Atlas Anticorpi </td></tr><tr><td> Donkey sec. anticorpo anti-coniglio Alexa Fluor 488, # A-21.206 o equivalente sec. immunofluorescenza </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> filtri cella 30 micron, 70 micron e 100 micron </td><td> BD Biosciences </td></tr><tr><td> Douncer vetro (40 ml) epestello &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax </td><td> IKA Germania </td></tr><tr><td> Disperdente strumento S25 N-18 G </td><td> IKA Germania </td></tr><tr><td> Centrifuga Beckman Avanti </td><td> Beckman Coulter </td></tr><tr><td> Provette Falcon da 15 ml e 50 ml </td><td> VWR </td></tr><tr><td> Tubi centrifuga Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotore oscillante libero </td><td> Beckman Coulter </td></tr><tr><td> Influx citometro </td><td> Beckman Coulter </td></tr><tr><td> Tubo Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificazione dei nuclei cardiomiociti è stato impegnativo in sezioni di tessuto, come la maggior parte delle strategie di contare solo su proteine ​​marker citoplasmatici 1. Eventi rari in miociti cardiaci quali la proliferazione e l&#39;apoptosi richiedono una identificazione accurata dei miociti cardiaci nuclei di analizzare il rinnovamento cellulare in omeostasi e in condizioni patologiche 2. Qui, forniamo un metodo per isolare nuclei cardiomyocyte dal tessuto post mortem di sedimentazione densità e immunomarcatura con anticorpi contro materiale pericentriolar 1 (PCM-1) e la successiva selezione citometria a flusso. Questa strategia permette una analisi un elevato throughput e l&#39;isolamento con il vantaggio di lavorare altrettanto bene su tessuti freschi e congelati materiale d&#39;archivio. Ciò rende possibile studiare materiale già raccolto in biobanche. Questa tecnica è applicabile e testato in una vasta gamma di specie e adatto a molteplici applicazioni a valle, come il carbonio-14 datazione 3, cell-cyCLE analisi 4, la visualizzazione di analoghi della timidina (ad esempio BrdU e IDU) 4, l&#39;analisi del trascrittoma ed epigenetici.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Nuclei cardiaca sono isolati tramite sedimentazione densità e immunolabeled con anticorpi contro materiale pericentriolar 1 (PCM-1) per identificare e ordinare nuclei cardiomiociti mediante citometria a flusso.

Isolamento dei Nuclei Cardiomyocyte dal post-mortem del tessuto

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

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16.9K Views.  University of Lund. Nuclei cardiaca sono isolati tramite sedimentazione densità e immunolabeled con anticorpi contro materiale pericentriolar 1 (PCM-1) per identificare e ordinare nuclei cardiomiociti mediante citometria a flusso.

Video: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolamento di isole umane da pazienti parzialmente Pancreatectomized

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

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Medicina

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Istituzione di umano epiteliali Enteroids e Colonoids dal tessuto intero e biopsia

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Strategie di campionamento e trattamento dei campioni di tessuto Biobank Porcine modelli biomedicale

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento di cellule deciduali umane primarie dalle membrane fetali del termine intempestivo della placenta

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Isolamento dei miociti atriale dai topi adulti

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Valutazione elettromeccanica dell'attività cardiomiocite ottogeneticamente modulata

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Modello di cardiomiociti ischemici e confronto video-based della contrazione cardiomiocite utilizzando cardiomiociti derivati da hiPSC

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolamento di adipociti vitali e frazione vascolare stromale dal tessuto adiposo viscerale umano adatto per l'analisi dell'RNA e la fenotipizzazione dei macrofagi

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...