Name: generation and isolation of cell cycle-arrested cells with complex karyotypes
Uploaded: 2018-04-13T13:00:00.000+00:00
Duration: 5 min 22 s
Description: Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

Questo lavoro è stato supportato in parte dal sostegno Istituto Koch (nucleo) Grant P30-CA 14051 dal National Cancer Institute, dal National Institutes of salute Grant (CA206157-22 e GM118066) e Curt marmo Cancer Research Fund di Angelika Amon. Angelika Amon è anche un investigatore di Howard Hughes Medical Institute e Glenn Foundation per la ricerca biomedica. S. S. è stata sostenuta dall'americano italiano cancro Foundation (AICF), di una borsa di studio nella ricerca sul cancro da azioni Marie Curie e l'associazione italiana per la ricerca cancro (AIRC) e di una borsa di ricerca cancro quinquennali KI. E.M. è stato sostenuto da una borsa di studio da Howard Hughes Medical Institute.

Condizione di cultura
Linea cellulare RPE-1 hTERT è stato coltivato in per volta Eagle Medium di Dulbecco (Tabella materiali) completate con 10% siero bovino fetale, 2 mM L-Glutammina e 100 U/ml di penicillina/streptomicina. Le cellule sono state incubate a 37 ° C con 5% CO2 in un ambiente umidificato. Il protocollo descritto di seguito è stato sviluppato utilizzando piatti 10 cm e tutti i dati tecnici riportati si riferiscono a quei piatti. Se utilizzando diversi piatti, scala verso l'alto o verso il basso di conseguenza.
1. sincronizzazione delle cellule RPE-1
<ol><li>Utilizzare appena scongelate cellule che non hanno subito più di 15 passaggi per tutti gli esperimenti. Il giorno 1, scongelare e dispensare ~2.5 - 5,0 x 10 celle di hTERT5 RPE-1 in un piatto di coltura del tessuto di 10 cm. Aggiungere 8 mL di terreno di coltura standard e incubare per una notte. Determinare il numero di cellulare utilizzando un contatore di cella standard (ad esempio un emocitometro). Nota: Densità cellulare si riferiscono a condizioni di crescita delle cellule RPE-1. Condizioni di crescita potrebbero variare tra diverse linee cellulari; si consiglia di regolare loro conseguenza.</li>
	<li>Il giorno 2, è necessario sincronizzare celle al bordo di G1/S ad aspirazione normale crescita medio e aggiungendo mezzo 8 mL contenente timidina 5 mM. Nota: Timidina concentrazione e durata del trattamento può variare in diverse linee cellulari. Si consiglia di eseguire esperimenti pilota per determinare le migliori condizioni per le righe di cella essere testato.</li>
	<li>Il giorno 3, 24 h dopo l'aggiunta della timidina medio (punto 1.2), rilasciare dal blocco di timidina aspirando medio e lavando le cellule tre volte con PBS 1X. Aggiungere 8 mL di medium di crescita regolare e restituire le piastre nell'incubatore.</li>
</ol>2. generazione di Aneuploid cellule tramite interferenza con l'attività della chinasi mitotica Mps1
<ol><li>Il giorno 3, 6 h dopo il rilascio dal blocco di timidina (passo 1.3; si tratta di circa 3-6 h prima celle immettere mitosi), aspirare medio, lavare una volta con PBS 1X e sostituire con mezzo contenente 500 nM reversine (l'inibitore Mps1). Nota: Le cellule RPE-1 immettere mitosi 9-12 h dopo timidina sbiaditura10,11. Tuttavia, la cinetica del ciclo cellulare variano tra differenti linee cellulari. Pertanto, è cruciale per determinare quelli cinetica in esperimenti pilota eseguendo analisi del ciclo cellulare di metodi consolidati (ad es., misurando l'analisi del DNA contenuto via FACS). Inoltre, la concentrazione di lavoro ottimale di Mps1 inibitore può variare tra linee cellulari. Precedenti esperimenti usando le cellule RPE1 hanno avuto successo usando reversine ad una concentrazione di lavoro di 500 nM10,11,12. Si consiglia di eseguire esperimenti pilota per determinare la concentrazione ottimale per la linea cellulare essere testato.</li>
	<li>Il giorno 4, 12 h dopo il trattamento reversine (circa 6 h dopo erano cellule in mitosi), aspirare reversine medio e lavare le cellule tre volte con PBS 1X. Aggiungere 8 mL di terreno di crescita regolare alla piastra e restituire le cellule nell'incubatore.</li>
</ol>3. rimozione di cellule aneuploidi in bicicletta e di arricchimento della popolazione ArCK
<ol><li>Il giorno 6, circa 72 h dopo celle immettere la prima mitosi (passaggio 2.2; si tratta di circa 66 h dopo reversine wash-out, che corrisponde a circa 2-3 cicli cellulari), aspirare medio, lavare una volta con PBS 1X e sostituire con mezzo contenente 300 nM nocodazole. Nota: Lavoro recente ha dimostrato che cellule RPE-1 che harboring i karyotypes aneuploidi sono genomically instabile e arrestano la loro proliferazione circa 2-3 cicli cellulari dopo i primi10di mitosi difettoso. Tuttavia, questa cinetica potrebbe variare tra diverse linee cellulari. Di conseguenza, è fondamentale eseguire esperimenti pilota per determinare il corretto timing per la rimozione di cellule aneuploidi in bicicletta.</li>
	<li>Il giorno 7, 12 h dopo il trattamento nocodazole, aspirare medio e aggiungere 3 mL di 1X PBS alla piastra. Scrollarsi di dosso le cellule mitotiche toccando il lato della piastra. Ruotare la piastra tra ogni rubinetto per consentire anche la rimozione di cellule mitotiche.</li>
	<li>Per garantire la completa rimozione di cellule mitotiche, ripetere il processo di shake-off per più turni (3 - 5 volte consigliati), aspirando e aggiungere 3 mL di 1X PBS tra ogni round.</li>
	<li>Dopo shake-off, delicatamente aspirare tutto il liquido che ha aderito il coperchio o il bordo della piastra. Brevemente, controllare la piastra con un microscopio a ingrandimento 10x. Assicurare che siano osservate alcune cellule mitotiche. Se più di cinque cellule mitotiche sono visti sotto obiettivo 10x, ripetere un altro giro di shake-off. Nota: Cellule mitotiche sembrerebbero arrotondate e possono essere parzialmente disconnesso dopo shake-off. È fondamentale garantire la completa rimozione di cellule mitotiche prima esposizione delle cellule al turno successivo del trattamento nocodazole. Non farlo può portare alla morte cellulare mitotica e/o causare la generazione di cellule tetraploidi.</li>
	<li>Dopo shake-off finale, lavare le cellule una volta con 5 mL di PBS 1X. Aggiungere 8 mL di terreno contenente 330 nM nocodazole nella piastra ed incubare per 12 h.</li>
	<li>Ripetere nocodazole trattamento/shake-off (passaggi 3.1-3.5) ogni 12 h fino a nessun cellule mitotic sono state osservate dopo incubazione. Di solito, sono consigliati 4-5 cicli di trattamento/shake-off. Nota: Le informazioni qui fornite si riferiscono alle cellule RPE-1. Il numero di giri del trattamento/shake-off potrebbe variare tra diverse linee cellulari. Per evitare l'esposizione prolungata e inutile a nocodazole è cruciale per determinare accuratamente, in un esperimento pilota, il numero di giri richiesto per rimozione completa ed efficiente di cellule aneuploidi in bicicletta.</li>
	<li>Dopo 4-5 giri di nocodazole trattamento/shake-off (passaggi 3.1-3.6), aggiungere 10 mL di medium di crescita regolare e incubare. Consentire alle cellule di riposare per 12-24 h prima dell'uso futuro di manipolazione o saggio. Nota: Dopo shake-off finale, cellule residue sulla piastra sono ciclo cellulare arrestato e, come recentemente dimostrato10, harbor karyotypes complessi. Queste cellule si riferiscono a come cellule di ArCK (arrestato con Karyotype complesso). Si consiglia di effettuare saggi sulle popolazioni di ArCK entro una settimana di isolamento.</li>
	<li>Facoltativamente, per valutare la percentuale di cellule arrestate con karyotypes complessi, raccogliere e contare le celle da ogni shake-off utilizzando un contatore di cella standard.</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg"> Figura 1: nel complesso schematico del metodo utilizzato per generare e isolare cellule aneuploidi con karyotype complesso (ArCK) e immagini rappresentative. (A) schematico di ArCK cellule protocollo di isolamento e di generazione. (B) immagini rappresentative delle cellule di hTERT RPE-1 in ogni fase del trattamento. L'ultimo pannello (evidenziato in rosso) rappresenta le cellule isolate di ArCK. Scala bar 100 µm. <a href="//cloudflare.jove.com/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
4. caratterizzazione della popolazione ArCK
<ol><li>Confermare successo isolamento della popolazione ArCK eseguendo i seguenti saggi.<ol>
<li>Asini della beta-galattosidasi che macchia (l'indicatore della senescenza) utilizzando un disponibili in commercio kit (Vedi tabella materiali).</li>
		<li>Misurare la secrezione di citochine pro-infiammatorie, quali CCL2, impiegando Kit commercialmente disponibili (Vedi tabella materiali).</li>
		<li>Confermare i livelli aumentati del ciclo cellulare inibitori p53, p21, p16 mediante Western Blot. Nota: Per controllare correttamente queste analisi, ArCK popolazioni devono essere confrontati con cellule ciclanti sia euploide e aneuploidi. Il primo è basso passaggio, hTERT RPE-1 selvaggio-tipo cellule in crescita esponenziale. Quest'ultimo può essere ottenuto inducendo mis-segregazione del cromosoma in RPE-1 hTERT e raccolta delle cellule 72 h dopo la prima mitosi aberrante. A tale scopo, seguire il protocollo descritto sopra dal passaggio 1.1 e raccolto le cellule destra prima del primo trattamento nocodazole (punto 3.1).</li>
	</ol>
</li>
	<li>Eseguire il sequenziamento di singola cellula per valutare il karyotype delle cellule arrestati. Nota: Il sequenziamento di cella singola è un metodo ben consolidato per determinare cambiamenti cromosomici e sub-cromosomici fra una popolazione di cellule al livello unicellulare13. Procedure dettagliate per come eseguire la sequenziazione di singola cellula possono essere trovate altrove10.</li>
</ol>

<ol>
	<li>Santaguida, S., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-+and+long-term+effects+of+chromosome+mis-segregation+and+aneuploidy.">Short- and long-term effects of chromosome mis-segregation and aneuploidy.</a> Nature Reviews Molecular Cell Biology. 16 (8), 473-485 (2015).</li><li>Pfau, S. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+instability+and+aneuploidy+in+cancer:+from+yeast+to+man.">Chromosomal instability and aneuploidy in cancer: from yeast to man.</a> EMBO reports. 13 (6), 515-527 (2012).</li><li>Gordon, D. J., Resio, B., Pellman, D. <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+aneuploidy+in+cancer.">Causes and consequences of aneuploidy in cancer.</a> Nature Reviews Genetics. 13 (3), 189-203 (2012).</li><li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boveri+revisited:+chromosomal+instability,+aneuploidy+and+tumorigenesis.">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nature Reviews Molecular Cell Biology. 10 (7), 478-487 (2009).</li><li>Meena, J. K., Cerutti, A., 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=Telomerase+abrogates+aneuploidy-induced+telomere+replication+stress,+senescence+and+cell+depletion.">Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.</a> The EMBO Journal. , 1-14 (2015).</li><li>Ohashi, A., Ohori, 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=Aneuploidy+generates+proteotoxic+stress+and+DNA+damage+concurrently+with+p53-mediated+post-mitotic+apoptosis+in+SAC-impaired+cells.">Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.</a> Nature Communications. 6, 7668 (2015).</li><li>Passerini, V., Ozeri-Galai, 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=The+presence+of+extra+chromosomes+leads+to+genomic+instability.">The presence of extra chromosomes leads to genomic instability.</a> Nature Communications. 7, 10754 (2016).</li><li>Sheltzer, J. M., Blank, H. 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=Aneuploidy+drives+genomic+instability+in+yeast.">Aneuploidy drives genomic instability in yeast.</a> Science. 333 (6045), 1026-1030 (2011).</li><li>Zhu, J., Pavelka, N., Bradford, W. D., Rancati, G., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotypic+determinants+of+chromosome+instability+in+aneuploid+budding+yeast.">Karyotypic determinants of chromosome instability in aneuploid budding yeast.</a> PLoS Genetics. 8 (5), e1002719 (2012).</li><li>Santaguida, S., Richardson, A., 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=Chromosome+Mis-segregation+Generates+Cell-Cycle-Arrested+Cells+with+Complex+Karyotypes+that+Are+Eliminated+by+the+Immune+System.">Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.</a> Developmental Cell. 41 (6), 638.e5-651.e5 (2017).</li><li>Santaguida, S., Vasile, E., White, E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy-induced+cellular+stresses+limit+autophagic+degradation.">Aneuploidy-induced cellular stresses limit autophagic degradation.</a> Genes &amp; Development. 29 (19), 2010-2021 (2015).</li><li>Santaguida, S., Tighe, A., D'Alise, A. M., Taylor, S. S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+role+of+MPS1+in+chromosome+biorientation+and+the+spindle+checkpoint+through+the+small+molecule+inhibitor+reversine.">Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.</a> The Journal of Cell Biology. 190 (1), 73-87 (2010).</li><li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genome+sequencing:+current+state+of+the+science.">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175-188 (2016).</li><li>L&#243;pez-Soto, A., Gonzalez, S., L&#243;pez-Larrea, C., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosurveillance+of+Malignant+Cells+with+Complex+Karyotypes.">Immunosurveillance of Malignant Cells with Complex Karyotypes.</a> Trends in cell biology. , 1-4 (2017).</li><li>Watson, E. V., Elledge, 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=Aneuploidy+Police+Detect+Chromosomal+Imbalance+Triggering+Immune+Crackdown!.">Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown!.</a> Trends in genetics: TIG. , 1-3 (2017).</li></ol>

Gli autori non hanno nulla a rivelare.

Questo nuovo metodo per generare e arricchire di cellule arrestato con karyotypes complessi (ArCK) consente lo studio delle cellule che hanno più del cromosoma guadagni o perdite e smettono di dividersi. Il metodo setup è stato progettato per facilitare l'isolamento di cellule ArCK in modo rapido e affidabile.
La fase più critica in questo test è rigorosamente il controllo timeline di trattamento farmacologico e, soprattutto, la rimozione di cellule aneuploidi in bicicletta. Per risultati ottimali, i tempi di trattamento nocodazole e shake-off sono di particolare importanza per garantire che le cellule ciclanti sono rimosse dalla piastra mentre sono ancora arrotondati e mitotica, impedendo la possibilità di morte cellulare mitotica o slittamento in G1, potenzialmente creando una popolazione tetraploide. Non è consigliabile che i tempi di shake-off devia più di due ore dall'incubazione consigliato 12-h nocodazole.
Gli studi futuri su ArCK cellule hanno il potenziale per facilitare una più profonda comprensione di come complesso karyotypes influisce sulla fisiologia cellulare. In particolare, uno studio recente ha dimostrato che le cellule del sistema immunitario sono in grado di interagire con e grilletto clearance immuni di ArCK cellule10. Il metodo qui descritto fornisce un ottimo punto di partenza per ulteriore caratterizzazione delle cellule di ArCK, compreso il chiarimento dei meccanismi molecolari che liquidazione immuni in cellule non trasformate e lo studio della trasformazione come oncogeni può ignorare questo meccanismo di sorveglianza, una questione cruciale nel campo14,15.

Errori nel processo di segregazione del cromosoma causare aneuploide, una condizione caratterizzata da un numero di cromosoma che non è un multiplo del aploide complemento1,2,3,4. Aneuploid karyotypes stress di replica di trigger che genera ulteriore instabilità genomica5,6,7,8,9,10, aumenta complessità del cariotipo e in ultima analisi porta ad arresto del ciclo cellulare di una sottopopolazione di cellule10. Lo scopo del metodo qui presentato è quello di generare e separare tali una sottopopolazione dal riciclaggio delle cellule. Utilizzando tecniche di coltura del tessuto economico, questo protocollo facilita l'isolamento e la caratterizzazione di cellule aneuploidi ciclo cellulare-arrestato con karyotypes complessi. Queste cellule sono denominate cellule ArCK (arrestato con Karyotype complesso) e le loro controparti in bicicletta euploide come controlli.
Questo protocollo è il primo ad essere istituito a tale scopo e permette l'isolamento e un ulteriore approfondimento delle linee di ArCK inclusi, ma non limitato a, loro indotta senescenza e il fenotipo secretivo senescenza-collegata (SASP), loro pro-infiammatorie caratteristiche e la loro capacità di impegnarsi con le cellule immunitarie. Il metodo presentato qui è stato sviluppato in cellule umane immortalizzate, non trasformate, ma non è ancora stato testato nelle linee del cancro. Alcune cellule trasformate possono essere insensibili all'arresto del ciclo cellulare a causa della soppressione di uno o più percorsi; di conseguenza, ulteriore convalida deve essere eseguita in altre linee cellulari.

<table><tbody><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle Medium)&amp;nbsp;</td><td>Thermofisher</td><td>11995-073</td><td></td></tr><tr><td>Thymidine</td><td>Sigma Aldrich</td><td>T1859</td><td></td></tr><tr><td>Reversine</td><td>Cayman Chemical Company</td><td>10004412</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma Aldrich</td><td>M1410</td><td></td></tr><tr><td>Anti-p53 antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Anti-p21 antibody</td><td>Santa Cruz</td><td>sc-6246</td><td></td></tr><tr><td>Anti-p16 antibody</td><td>BD</td><td>554079</td><td></td></tr><tr><td>Senescence b-Galactosidase Staining Kit</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9860</td><td></td></tr><tr><td>Proteome Profiler Human Cytokine Array Kit&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>ARY005B</td><td></td></tr></tbody></table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

Mis-segregazione del cromosoma conduce ad aneuploide, una condizione in cui cellule del porto un numero cromosomico squilibrato. Parecchie linee di prova indicano fortemente che aneuploide innesca instabilità genomica, in ultima analisi, generare cellule con karyotypes complessi che arrestano la loro proliferazione. Isolamento e caratterizzazione di cellule che harboring i karyotypes complessi sono cruciali per studiare l'impatto di un numero di cromosoma squilibrato sulla fisiologia cellulare. Ad oggi, non sono stati stabiliti metodi per isolare in modo affidabile tali cellule aneuploidi. Questo documento fornisce un protocollo per l'arricchimento e l'analisi di cellule aneuploidi con karyotypes complessi utilizzando tecniche di coltura del tessuto standard, poco costoso. Questo protocollo può essere usato per analizzare le diverse caratteristiche di cellule aneuploidi con karyotypes complessi inclusi indotta senescenza-collegata di fenotipo secretivo, proprietà pro-infiammatorie e capacità di interagire con le cellule immunitarie. Perché le cellule tumorali spesso porto gli squilibri nel numero dei cromosomi, è fondamentale decifrare come aneuploide impatti fisiologia cellulare in cellule normali, con l'obiettivo finale di scoprire entrambi suoi pro - e anti - tumorigenic effetti.

Questo metodo utilizza un sistema di coltura di tessuti in vitro per isolare cellule aneuploidi con karyotypes complessi che arrestano la loro proliferazione. Questa popolazione viene riferita come celle di ArCK (arrestato con Karyotypes complessi). Figura 1A Mostra lo schema dell'esperimento. Cellule di hTERT ciclismo RPE-1 wild-type sono sincronizzate al bordo di G1/S con timidina e trattate con un inibitore di Mps1 per indurre mis-segregazione del cromosoma. Il nocodazole veleno dell'alberino è aggiunto alle cellule 72h dopo induzione di mis-segregazione del cromosoma. 12 h dopo il trattamento nocodazole, le cellule aneuploidi che sono ancora in grado di proliferare immettere mitosi e sono intrappolati in questa fase del ciclo cellulare a causa dell'interferenza con la polimerizzazione dei microtubuli. Questi intrappolato le cellule vengono poi rimossi facilmente dal delicato shake-off. Il processo di shake-off è ripetuta 3 - 5 volte per garantire la completa rimozione del riciclaggio delle cellule. Figura 1B Mostra le immagini rappresentative delle cellule RPE-1 in ogni fase del trattamento.
ArCK celle visualizzare i livelli aumentati di inibitori del ciclo cellulare, quali p53, p21 e p16 rispetto al euploide e aneuploid riciclaggio delle cellule, come mostrato nella Figura 2A. Inoltre, la Figura 2B Mostra positiva β-galattosidasi macchiatura in cellule di ArCK rispetto alle cellule di controllo euploide, un indicatore ben consolidato per la senescenza cellulare.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg"> Figura 2: risultati rappresentativi che utilizzano cellule di ArCK. (A) valutazione dei livelli dell'inibitore ciclo cellulare in euploide aneuploid ciclismo e cellule di ArCK macchia occidentale. I livelli di p53, p21 e p16 sono stati determinati mediante analisi western blot. Actina servito come un controllo di carico. (B) β-galattosidasi macchiatura valutazione del livello di senescenza in cellule di ArCK. Attività di β-galattosidasi senescenza-collegata (β-gallone) è stata determinata in euploide e cellule di ArCK. Scala bar 100 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
Questo arresto del ciclo cellulare è una caratteristica prominente di cellule aneuploidi con karyotypes complessi, come indicato dall'analisi del cariotipo rappresentativo da unicellulare sequenziamento in Figura 3, in cui le cellule ArCK visualizzano multipli, random del cromosoma guadagni e perdite. Questa scoperta è in pieno accordo con precedenti rapporti10.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg"> Figura 3: rappresentanza unicellulare sequenziamento delle cellule di ArCK. Segmentazione diagrammi che mostrano il karyotype del rappresentante ArCK sei celle. Segmentazione grafici mostrano il numero di copie di tutti i cromosomi da 1 a X rispetto a un riferimento euploide su una scala di2 log (tipo selvaggio RPE-1 è una linea di cellula diploide di origine femminile). Cromosoma guadagni sono evidenziati in rosso, perdite di cromosoma in verde. <a href="https://cloudflare.jove.com/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Aneuploide conduce all'instabilità del genoma, che alla fine produce le cellule ciclo cellulare-arrestato con karyotypes complessi. Questo documento fornisce un metodo semplice e conveniente per isolare cellule aneuploidi con karyotypes complessi che non smettono di dividersi.

Generazione e l'isolamento delle cellule ciclo cellulare-arrestato con Karyotypes complessi

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

generation-isolation-cell-cycle-arrested-cells-with-complex

Research

JoVE Journal

Cancer Research

10.4K Views.  Massachusetts Institute of Technology. Aneuploide conduce all'instabilità del genoma, che alla fine produce le cellule ciclo cellulare-arrestato con karyotypes complessi. Questo documento fornisce un metodo semplice e conveniente per isolare cellule aneuploidi con karyotypes complessi che non smettono di dividersi.

Video: Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Studiare l&#39;espressione del gene regolamentata mediante ciclo cellulare da due protocolli complementari di sincronizzazione delle cellule

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Ricerca

Genetica

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

Preparazione delle cellule di leucemia linfoblastica acuta primaria nel ciclo cellulare diverse fasi di classificazione mediante veicolo fluido centrifuga

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Ricerca sul cancro

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Lo studio di RNA interattori della chinasi proteica RNA-attivato durante il ciclo cellulare dei mammiferi

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Biochimica

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Live Cell Imaging della segregazione cromosomica durante la mitosi

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Biologia dello sviluppo

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Preparazione Stendere cromosomiche di cellule staminali embrionali umane per cariotipo

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Biologia

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Cella di misura Kinetics progressione del ciclo con la marcatura metabolica e citometria a flusso

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Cromosoma Preparazione da cellule in coltura

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Un Free Cell Assay per studiare cromatina decondensazione alla fine del mitosi

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...