The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).

Il protocollo qui descritto utilizza la linea cellulare linfoblastica acuta leucemia NALM6 ma può essere applicato ad altri tipi di cellule. 1. Soluzioni e reagenti <ol><li> Completa RPMI <ol><li> Aggiungere 56 ml di siero fetale bovino (FCS) e 5,5 ml di 200 mM di L-glutammina in un flacone da 500 ml di mezzo RPMI-1640. </li></ol></li><li> BrdU Stock Solution <ol><li> Preparare 32.5 BrdU mm (10 mg / ml) in Dulbecco tampone fosfato (DPBS). </li></ol></li><li> BrdU completo RPMI <ol><li> Aggiungere 6,2 ml di soluzione madre BrdU per 10 ml di RPMI completo. </li></ol></li><li> DNase Soluzione <ol><li> Preparare 1 mg DNase / ml in DPBS. </li></ol></li><li> Colorazione Buffer <ol><li> Preparare 3% FCS inattivato al calore e 0,09% di sodio azide in DPBS. </li></ol></li><li> Vedi Materiali Lista per le definizioni di Fixation Buffer, Permeabilization tampone e tampone di lavaggio. </li></ol> 2. Cellule NoTE: Le cellule non sono state coltivate per oltre 6 mesi. Questo metodo è direttamente adattabile a qualsiasi linea cellulare non aderente con adattamenti densità cellulare e coltura. Utilizzare le cellule che stanno crescendo in modo esponenziale a inizio dell&#39;esperimento. <ol><li> Mantenere le cellule NALM6 in T-75 fiasche di coltura in RPMI completo. Eseguire tutte le operazioni in condizioni sterili utilizzando una classe II biosicurezza Cabinet. <ol><li> Mantenere le cellule NALM6 tra 1-2 x 10 6 cellule per ml dividendo cultura tre volte alla settimana. </li><li> Incubare a 37 ° C in 5% CO 2 in aria. </li></ol></li></ol> 3. Pulse Etichettatura di Celle con BrdU ATTENZIONE: Maneggiare con cura BrdU in quanto è un potenziale mutageno e teratogeno. <ol><li> Cellule centrifugare a 150 xg per 5 min. Nota: Trasferimento di cellule in mezzi freschi migliora la riproducibilità dei risultati. </li><li> Eseguire un numero di celle e risospendere le cellule in RPMI completo a 2 x 10 6 cells / ml. </li><li> Diluire le cellule in 1 2 con BrdU completo RPMI producendo una concentrazione cellulare finale di 1 x 10 6 cellule / ml. </li><li> Incubare a 37 ° C con 5% di CO 2 per 45 minuti, poi diluito 1 a 10 celle con RPMI completo. Cellule centrifugare a 150 xg per 5 minuti e con attenzione scartano tutto il surnatante. </li><li> Risospendere le cellule in un piccolo volume (~ 100 ml) di RPMI completo, eseguire un conteggio delle cellule e regolare per 1 x 10 6 cellule / ml. </li><li> Pipettare 1 ml di cellule nei pozzetti di una piastra ben 48. Pipettare 1 ml di DPBS in qualsiasi pozzetti non occupate per ottenere risultati più riproducibili. </li><li> Incubare a 37 ° C in 5% CO 2 in aria per timepoints desiderati, qui 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, e 24 ore. Nota: La lunghezza del tempo dipenderà da ciò che il disegno sperimentale mira a misurare. </li><li> Trasferire tutte le cellule in tubi FACS con una pipetta. Sciacquare la sequenza bene con 1 ml volumes di PBS per un volume totale finale di 5 ml. </li><li> Centrifugare a 150 xg per 5 minuti e rimuovere con attenzione tutto il surnatante. Le cellule sono pronte per la colorazione, eseguire questa (sezione 4) immediatamente. </li></ol> Colorazione 4. cellulare Nota: Se è richiesta superficie colorazione di cellule eseguirla prima del fissaggio, in modo che le cellule sono conservati a 4 ° C per tutta. <ol><li> Risospendere le cellule in 100 ml di tampone colorazione (per facoltativo colorazione della superficie, aggiungere il volume consigliato di anticorpi di superficie antigeni e incubare per 30 minuti a 4 ° C). </li><li> Aggiungere 1 ml di tampone colorazione, centrifugare per 5 min a 150 xg ed eliminare il surnatante. Nota: l&#39;anticorpo specifico, la concentrazione, il tempo di incubazione ecc varierà a seconda degli obiettivi sperimentali specifici. </li><li> Fissazione e permeabilizzazione <ol><li> Risospendere le cellule in 100 microlitri di tampone di fissaggio e incubare per 15 min a temperatura ambiente. </li><li> Aggiungere 1 ml of tampone di lavaggio, centrifugare per 5 min a 150 xg ed eliminare il surnatante. </li><li> Risospendere le cellule in 100 microlitri di tampone permeabilizzazione e incubare le cellule per 10 min in ghiaccio. </li><li> Aggiungere 1 ml di tampone di lavaggio, centrifuga per 5 minuti a 150 xg, e scartare il surnatante. </li><li> Risospendere le cellule in 100 microlitri di tampone di fissaggio per tubi e incubare per 5 min a temperatura ambiente. </li><li> Aggiungere 1 ml di tampone di lavaggio, centrifuga per 5 minuti a 150 xg, e scartare il surnatante. Nota: Il protocollo può essere messo in pausa qui, se necessario. Le cellule fissate sono stabili per diversi giorni a 4 ° C se risospeso in tampone colorazione. Rimuovere il buffer di colorazione seguente centrifugazione prima di procedere. </li></ol></li><li> DNase Trattamento <ol><li> Risospendere le cellule in 100 microlitri di soluzione di DNasi (30 microgrammi di DNasi / 10 6 cellule) e incubare le cellule per 1 ora a 37 ° C. </li><li> Aggiungere 1 ml di tampone di lavaggio, centrifugare a 150 xg per 5 minuti e scartare il surnatante. </li&gt; </ol></li><li> Anticorpi colorazione Nota: colorazione di marcatori intracellulari diversi BrdU può essere eseguita contemporaneamente alla colorazione BrdU. <ol><li> IMPORTANTE: Preparare controlli di compensazione costituiti da cellule non colorati e cellule marcate con ogni singolo fluorocromo. Idealmente, utilizzare gli stessi anticorpi per i controlli di compensazione come quelle utilizzate nei tubi sperimentali. Tuttavia, se ciò non è fattibile, anticorpi sostituto antigeni altamente espressi coniugati allo stesso fluorocromo. </li><li> Risospendere le cellule in 50 ml di tampone di lavaggio e aggiungere 1 ml / 10 6 cellule di anticorpi BrdU. Nota: anticorpi coniugati direttamente ad altri antigeni intracellulari specifici possono anche essere aggiunti. NOTA:. Gli anticorpi anti istone H3 fosforilata in Ser10 possono essere utilizzati per discriminare tra cellule in G2 e M, istone H3 è fosforilata in Ser10 durante la mitosi 10 Anticorpi anti cdc2 fosforilati su Tyr15 può essere utilizzato per rilevare le cellule che HAVe impegnata per mitosi. 11 </li><li> Incubare le cellule per 20 minuti a temperatura ambiente. </li><li> Aggiungere 1 ml di tampone di lavaggio, le cellule centrifugare a 150 xg per 5 minuti e scartare il surnatante. </li></ol></li></ol><ol start="6"><li> DNA Stain per l&#39;analisi del ciclo cellulare <ol><li> Allentare pellet e aggiungere 20 ml di soluzione di 7-AAD (0,25 mg). Nota: è fondamentale utilizzare una quantità costante di 7-AAD / cell. </li><li> Risospendere le cellule in 1 ml di tampone di colorazione. </li></ol></li></ol> 5. Raccolta di Citometria a Flusso di dati Nota: La macchina richiesta dipenderà dal numero e natura dei fluorocromi usati. <ol><li> Raccogliere i seguenti parametri: FSC-A, SSC-A, FSC-H (FSC-W possono essere utilizzati al posto di FSC-H) e 7-AAD fluorescenza su una scala lineare. Raccogliere il canale APC su una scala logaritmica. Raccogliere eventuali canali aggiuntivi necessari per la valutazione della superficie o interne etichette utilizzando una scala logaritmica. </li><li> Eseguire comcompensazione dei segnali in spettri di emissione osservata tra diversi fluorocromi prima di analizzare i campioni si sovrappongono. Nota: La maggior parte dei citofluorimetri eseguirà automaticamente. </li><li> Raccogliere almeno 10.000 eventi per ciascun campione. </li></ol> 6. Analisi di citometria a flusso di dati Nota: FlowJo stato utilizzato in questo studio per citometria di flusso possono essere utilizzati anche l&#39;analisi dei dati, ma altri pacchetti software. La strategia di gating è illustrato nella figura 1. <ol><li> Identificare la popolazione di cellule vitali con i parametri FSC-A e SSC-A. </li><li> All&#39;interno di questa popolazione escludere doppietti e aggregati utilizzando FSC-A e FSC-H (FSC-W possono anche essere usato qui). </li><li> All&#39;interno di questa popolazione impostare un diagramma a punti con 7-AAD sulla xe BrdU-APC sul y. </li></ol><img alt="Figura 1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> Figura 1: strategia di gating pa sinistra.nel: cellule ungated vengono visualizzati su un FCS-A e SSC-A dot plot. La popolazione di cellule vitali è identificata dalla porta mostrato. Pannello centrale: le cellule gated dal pannello a sinistra vengono visualizzati su un FSC A vs. FSC-H dot plot (FSC-W può essere usato al posto di altezza). Farsetti e aggregati sono identificati, ed esclusi dalla porta mostrato. A destra: le cellule gated dalla data di esclusione doppietto nel pannello centrale vengono visualizzati su un 7-AAD vs. APC-A diagramma a punti. L&#39;anticorpo è marcato con BrdU APC consentono di identificare le cellule che hanno incorporato BrdU durante l&#39;etichettatura impulso. 7-AAD fornisce informazioni sul contenuto di DNA. La porta superiore definisce cellule positive per BrdU e quindi in fase S al tempo dell&#39;impulso BrdU, porta inferiore sinistra, cellule in G 0/1 e la porta inferiore destro quelli in G 2 / M. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">Fare click qui per visualizzare un versione più grande di questa figura.</a> <ol start="4"><li> Cell Cycle A<html> nalisi <ol><li> Aprire il primo file di dati porta e sulle cellule del cancello esclusione doppietto. </li><li> Analizzare questa popolazione per la distribuzione del ciclo cellulare (che si trova sotto le piattaforme di software Flojo) e utilizzare il modello Dean-Jett-Fox. </li><li> Ottenere le posizioni del G 0/1 e G 2 / M vette utilizzando creare cancelli. </li><li> Porta sul BrdU cellule positive e soggetti a questa popolazione stessa analisi del ciclo cellulare. </li><li> Fornire le posizioni per la G 0/1 e G 2 / M picchi applicando le stesse porte da creare porte e impostare vincoli (utilizzando le porte create) per le posizioni del G 0/1 e G 2 / M picchi. Questo è illustrato nei primi 2 pannelli di figura 2. Nota: altri software può essere utilizzato anche per analizzare i dati e le istruzioni varierebbe di conseguenza. </li></ol></li></ol>840 / 52840fig2highres.jpg &quot;width =&quot; 700 &quot;/&gt; Figura 2:. Cell Cycle Progression Il primo pannello (tutte le celle) è recintato sulla popolazione di cellule definito dalla porta esclusione farsetto. Questa popolazione è stata visualizzata in un istogramma con 7-AAD sulla X-asse. Il picco della G 0/1 picco è indicata dalla freccia sotto dell&#39;asse. In pannelli successivi cellule positive BrdU sono stati gated come mostrato in Figura 1. Il valore per la posizione G 0/1 ottenuto quando gating del cancello esclusione doppietto è applicato alle cellule BrdU positive gated nel software ciclo cellulare FlowJo. Ogni pannello posteriore sia stato gating sulla popolazione positiva BrdU come mostrato nella figura 1 e la posizione del G 0/1 picco in base al valore ottenuto quando si analizza l&#39;intera popolazione come mostrato nelle prime due pannelli. Utilizzando la frazione negativo BrdU per identificare la posizione della popolazione G 0/1 per le cellule positive BrdU nella stessa SAMPle controlla per eventuali lievi differenze nell&#39;intensità della macchia DNA tra i campioni. Il numero indicato su ogni pannello rappresenta il tempo da quando l&#39;impulso BrdU conclusa. Le fasi del ciclo cellulare calcolati sono mostrati in verde ombreggiato. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

<ol>
	<li>Banfalvi, G., Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Mol Biol. 761, 1-23 (2011).</li><li>Sakaue-Sawano, 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=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132, 487-498 (2008).</li><li>Harper, J. W., 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=The+DNA+damage+response:+ten+years+after.">The DNA damage response: ten years after.</a> Molecular Cell. 28, 739-745 (2007).</li><li>Latt, S. A., George, Y. S., Gray, J. W. <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+analysis+of+bromodeoxyuridine-substituted+cells+stained+with+33258+Hoechst.">Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.</a> The Journal of Histochemistry and Cytochemistry. 25, 927-934 (1977).</li><li>Gratzner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+to+5-bromo-+and+5-iododeoxyuridine:+A+new+reagent+for+detection+of+DNA+replication.">Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication.</a> Science. 218, 474-475 (1982).</li><li>Carayon, P., Bord, A. <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+DNA-replicating+lymphocyte+subsets+using+a+new+method+to+label+the+bromo-deoxyuridine+incorporated+into+the+DNA.">Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA.</a> Journal of Immunological Methods. 147, 225-230 (1992).</li><li>Gonchoroff, N. J., 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=S-phase+detection+with+an+antibody+to+bromodeoxyuridine.+Role+of+DNase+pretreatment.">S-phase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment.</a> Journal of Immunological Methods. 93, 97-101 (1986).</li><li>Rabinovitch, P. S., Torres, R. M., Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+cell+cycle+analysis+and+two-color+surface+immunofluorescence+using+7-amino-actinomycin+D+and+single+laser+excitation:+applications+to+study+of+cell+activation+and+the+cell+cycle+of+murine+Ly-1+B+cells.">Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells.</a> Journal of Immunology. 136, 2769-2775 (1986).</li><li>Saunders, P. O., 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=RAD001+(everolimus)+induces+dose-dependent+changes+to+cell+cycle+regulation+and+modifies+the+cell+cycle+response+to+vincristine.">RAD001 (everolimus) induces dose-dependent changes to cell cycle regulation and modifies the cell cycle response to vincristine.</a> Oncogene. 32, 4789-4797 (2013).</li><li>Hendzel, M. J., 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=Mitosis-specific+phosphorylation+of+histone+H3+initiates+primarily+within+pericentromeric+heterochromatin+during+G2+and+spreads+in+an+ordered+fashion+coincident+with+mitotic+chromosome+condensation.">Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.</a> Chromosoma. 106, 348-360 (1997).</li><li>Draetta, G., Beach, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+cdc2+protein+kinase+during+mitosis+in+human+cells:+cell+cycle-dependent+phosphorylation+and+subunit+rearrangement.">Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement.</a> Cell. 54, 17-26 (1988).</li><li>Saunders, P. O., 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=RAD001+(everolimus)+induces+dose-dependent+changes+to+cell+cycle+regulation+and+modifies+the+cell+cycle+response+to+vincristine.">RAD001 (everolimus) induces dose-dependent changes to cell cycle regulation and modifies the cell cycle response to vincristine.</a> Oncogene. , (2012).</li><li>Knudsen, R. C., Ahmed, A. A., Sell, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microassay+for+lymphotoxin+using+microculture+plates+and+the+multiple+automated+sample+harvester.">An in vitro microassay for lymphotoxin using microculture plates and the multiple automated sample harvester.</a> Journal of Immunological Methods. 5, 55-63 (1974).</li><li>Beck, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+kinetics+of+perturbed+cell+populations+determined+by+the+bromodeoxyuridine-33258+technique:+radiotoxic+effects+of+incorporated+[3H]thymidine.">Proliferation kinetics of perturbed cell populations determined by the bromodeoxyuridine-33258 technique: radiotoxic effects of incorporated [3H]thymidine.</a> Cytometry. 2, 170-174 (1981).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Collins, J. M., Berry, D. E., Bagwell, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+rates+of+DNA+synthesis+during+the+S+phase+of+log+phase+HeLa+S3,+WI-38,+and+2RA+cells.">Different rates of DNA synthesis during the S phase of log phase HeLa S3, WI-38, and 2RA cells.</a> Journal of Biological Chemistry. 255, 3585-3590 (1980).</li><li>Schwarting, R., Gerdes, J., Niehus, J., Jaeschke, L., Stein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+growth+fraction+in+cell+suspensions+by+flow+cytometry+using+the+monoclonal+antibody+Ki-67.">Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67.</a> Journal of Immunological Methods. 90, 65-70 (1986).</li><li>Danova, 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=Cell+cycle-related+proteins:+a+flow+cytofluorometric+study+in+human+tumors.">Cell cycle-related proteins: a flow cytofluorometric study in human tumors.</a> Biology of the Cell. 64, 23-28 (1988).</li><li>Juan, G., 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=Histone+H3+phosphorylation+and+expression+of+cyclins+A+and+B1+measured+in+individual+cells+during+their+progression+through+G2+and+mitosis.">Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis.</a> Cytometry. 32, 71-77 (1998).</li><li>Langan, T. J., Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+mammalian+cell+cultures+by+serum+deprivation.">Synchronization of mammalian cell cultures by serum deprivation.</a> Methods in Molecular Biology. 761, 75-83 (2011).</li><li>Kues, W. 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=Cell+cycle+synchronization+of+porcine+fetal+fibroblasts:+effects+of+serum+deprivation+and+reversible+cell+cycle+inhibitors.">Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors.</a> Biology of Reproduction. 62, 412-419 (2000).</li><li>Urbani, L., Sherwood, S. W., Schimke, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+nuclear+and+cytoplasmic+cell+cycle+progression+by+drugs+employed+in+cell+synchronization.">Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization.</a> Experimental Cell Research. 219, 159-168 (1995).</li></ol>

The authors have nothing to disclose.

La capacità di analizzare il ciclo cellulare è importante per la comprensione della biologia del cancro e il meccanismo di azione di entrambi i farmaci e geni che influenzano la proliferazione e la crescita cellulare. Mentre ci sono una moltitudine di saggi che misurano riferito proliferazione cellulare, la maggioranza solo fornire una misura che indica le cellule numerici presenti. Questi includono test che misurano il numero di cellulare per la visualizzazione diretta e il conteggio, l&#39;attività metabolica o la ...

La valutazione delle caratteristiche del ciclo cellulare e cambiamenti che si verificano nelle cellule durante la progressione del ciclo cellulare è fondamentale per capire molti aspetti della biologia, in particolare biologia del cancro. Molti agenti di sviluppo per il trattamento di tumori maligni hanno effetti profondi sulla progressione del ciclo cellulare o indurre la morte cellulare per ciclo cellulare dipendente-meccanismi. Per studiare la dinamica del ciclo cellulare o cellule in una particolare fase del ciclo cellulare, si è soliti sincronizzare cellule. Tuttavia metodi di sincronizzazione possono avere effetti negativi sulle cellule in fase di studio, potenzialmente confondere i risultati ottenuti. 1 analisi Recentemente l&#39;uso di proteine ​​con tag fluorescenza che sono presenti solo in determinati fasi del ciclo di cellule hanno permesso di progressione del ciclo cellulare in cellule singole nel tempo 2, tuttavia le cellule da studiare bisogno di essere manipolato geneticamente per esprimere queste proteine ​​taggati, limitando il loro utilizzo ai sistemi in cui questo può essere lettoily raggiunto. Il ciclo cellulare è costituito da due fasi attive: la fase di sintesi (S), in cui il DNA si replica e la mitosi (M), dove la divisione cellulare si svolge. Queste fasi sono separate da tre fasi gap, G 0, G 1 e G 2. G 0 o quiescenza, è una fase di riposo in cui la cellula ha lasciato il ciclo, G 1 è dove le cellule aumentano di dimensione prima replicazione del DNA e G 2 dove la crescita cellulare continua tra il completamento della replicazione del DNA ma prima divisione cellulare. La progressione attraverso il ciclo cellulare è controllato da una serie di punti di controllo. Il G 1 checkpoint si attiva quando le condizioni ambientali non sono favorevoli della sintesi del DNA e impedisce l&#39;entrata in fase S. La fase di checkpoint o ritardo intra-S possono essere attivati ​​da danni al DNA che può provocare forche replica stallo. Durante G 2 la fedeltà del DNA replicato è confermata e se il danno è rilevato poi il G 2 </sub&gt; checkpoint è attivata permettendo la riparazione del DNA prima di divisione cellulare. Un punto di controllo finale durante la mitosi assicura che cromatidi sono state allineate correttamente la piastra mitotico in modo che la divisione cellulare può essere completata con successo. 3 L&#39;attivazione di questi punti di controllo è comunemente usato per sincronizzare popolazioni cellulari. Checkpoint del ciclo cellulare possono essere attivati ​​da una serie di fattori, ma in biologia del cancro il più comune è il rilevamento dei danni al DNA. La risposta al danno al DNA è iniziata dalla PI3-chinasi-like chinasi telangiectasia atassia e RAD3 connessi (ATR) e atassia telangiectasia mutato (ATM) che attivano chinasi effettori a valle Chk1 e CHK2 rispettivamente. 3 una serie di eventi attiva Chk1 compresi stallo forche replica, legami crociati DNA, e danni radiazione ultravioletta mentre Chk2 è attivata principalmente da rotture del doppio filamento. Il metodo usuale per studiare l&#39;effetto di mutate condizioni della lunghezza del ciclo cellulare is per sincronizzare le cellule in una particolare fase del ciclo cellulare. 1 Ciò può essere ottenuto mediante diversi metodi. Le celle possono essere fisicamente separate in base alle dimensioni, densità, dispersione laterale (granularità), e marcatori di espressione di superficie cellulare. Più in concreto, le cellule possono essere sincronizzati con mezzi chimici. Diversi agenti come timidina, citosina arabinoside e idrossiurea possono essere utilizzati per inibire la sintesi del DNA nella fase S del ciclo cellulare risultante in un accumulo di cellule nella fase S che continuano ciclismo dopo che gli agenti sono rimossi. Le cellule trattate con nocodazole, che impedisce la formazione del fuso mitotico, l&#39;arresto a G 2 - o M-fase contenuto di DNA. Eliminazione di siero dai risultati medi cultura in accumulo di cellule in G 0 fase. Tale aggiunta dei nutrienti nei siero cultura ri-avvia il normale bicicletta delle cellule. Tuttavia, tutti questi metodi di sincronizzazione interferisce con il ciclismo normale e la crescita delle cellule e possono Result significativa morte cellulare. La sincronizzazione delle cellule di leucemia linfoblastica acuta è particolarmente impegnativo e queste cellule non sono suscettibili di manipolazione genetica. Il metodo qui descritto consente di stimare le dinamiche del ciclo cellulare e lo studio delle cellule in particolari fasi del ciclo cellulare senza sincronizzazione tradizionale o modificazione genetica. Questo metodo può anche essere utile per altri tipi di cellule in cui la modificazione genetica e procedure di sincronizzazione tradizionali non sono facilmente ottenibili. Il metodo si basa sull&#39;uso consolidata di bromodeossiuridina (BrdU), che ha un impatto sulla crescita a breve termine e la proliferazione delle cellule. 4 protocolli BrdU fondazione sfruttano l&#39;incorporazione di BrdU nel DNA di nuova sintesi in fase S . Questo segna definitivamente le cellule come dopo essere stato in fase S durante l&#39;esposizione BrdU. Questa popolazione può essere identificato in momenti successivi con la colorazione per BrdU Incorporzione e quindi agiscono come una popolazione sincronizzato che può essere seguito e valutati nel corso del tempo permettendo lo studio degli effetti dei farmaci sul transito del ciclo cellulare. BrdU deve essere esposto prima della colorazione degli anticorpi, di solito raggiunti per DNasi o trattamento con l&#39;acido. 6,7 citometria a flusso per rilevare BrdU incorporata consente l&#39;inserimento di altri marker. La più importante è l&#39;uso di coloranti per misurare il contenuto di DNA, consentendo la valutazione del ciclo cellulare distribuzione di fase delle cellule che erano in fase S all&#39;inizio dello studio. 8 Inoltre aggiuntivi antigeni di superficie o intracellulari possono essere studiate. 9 Questi possono riguardare eventi del ciclo cellulare, come Ki67 o funzioni cellulari per apparentemente non correlate, come marcatori di apoptosi, come spaccati caspasi-3. Le potenziali applicazioni sono limitate dalla fantasia del ricercatore.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>APC BrdU Flow Kit</td>
			<td>BD Biosciences</td>
			<td>552598</td>
			<td>Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm 
			Buffer (referred to as Fixation Buffer)</td>
		</tr>
		<tr>
			<td>BD Cytoperm Permeabilization Buffer Plus</td>
			<td>BD Biosciences</td>
			<td>561651</td>
			<td>Referred to as Permeabilization buffer</td>
		</tr>
		<tr>
			<td>BD Perm/Wash Buffer</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Referred to as Wash buffer</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>D-4513</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 12 x 75-mm FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>554656</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR FORTESSA flow cytometer</td>
			<td>BD Biosciences</td>
			<td>FORTESSA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman</td>
			<td>Gilson</td>
			<td>P2, P20, P100, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o L-Gln 500 ml</td>
			<td>Lonza</td>
			<td>12-167F</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>FisherBiotec</td>
			<td>FBS-7100113</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-2&#8242;-deoxyuridine</td>
			<td>Sigma</td>
			<td>B5002-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon TC 150cm2 vented Flasks</td>
			<td>BD Biosciences</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 25mL</td>
			<td>Greiner</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 200&#181;L</td>
			<td>Interpath</td>
			<td>24700</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 1mL</td>
			<td>Interpath</td>
			<td>24800</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Spintron</td>
			<td>GT-175R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Binder</td>
			<td>C 150</td>
			<td></td>
		</tr>
		<tr>
			<td>AF488 anti-Histone H3 Phospho (Ser10) Antibody</td>
			<td>Cell Signalling</td>
			<td>9708S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td>2197S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk1 (Ser345) (133D3) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td><a name="RANGE!C23">2348S</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NALM6&#160;</td>
			<td>DSMZ</td>
			<td>ACC-128</td>
			<td></td>
		</tr>
	</tbody>
</table>

temporal tracking of cell cycle progression using flow cytometry without the need for synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

Questo metodo può essere utilizzato per ottenere una serie di informazioni. Alcune applicazioni sono elencate qui. Valutazione della durata del ciclo cellulare Per determinare il tempo necessario per le cellule di transito attraverso il ciclo cellulare, le cellule vengono raccolte in vari momenti seguenti dell&#39;impulso BrdU. Gli intervalli tra le valutazioni possono essere adattati alle particolari cellule essere analizzati. Linee cellulari ematopoietiche son...

Watch this Scientific Journal Video about Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization at JoVE.com

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization | Biology | Video

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Questo protocollo descrive l&#39;uso di bromodeossiuridina (BrdU) uptake per consentire il monitoraggio temporale di cellule che erano in fase S in un determinato momento. L&#39;aggiunta di coloranti DNA e l&#39;etichettatura degli anticorpi facilita l&#39;analisi dettagliata del destino delle cellule in fase S in tempi successivi.

Monitoraggio temporale del ciclo cellulare progressione citometria a flusso, senza la necessità di sincronizzazione

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

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Biology

Cellular Processes

Cell Cycle and Division

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19.0K Views.  Westmead Millennium Institute for Medical Research and University of Sydney.  Questo protocollo descrive l'uso di bromodeossiuridina (BrdU) uptake per consentire il monitoraggio temporale di cellule che erano in fase S in un determinato momento. L'aggiunta di coloranti DNA e l'etichettatura degli anticorpi facilita l'analisi dettagliata del destino delle cellule in fase S in tempi successivi. 

Video: Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificazione Citometria a flusso di cellule di topo Meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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Biologia

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 ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Cella di rilevamento utilizzando proteine ​​Photoconvertible durante lo sviluppo Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Tecniche per l&#39;Analisi dei extracellulare vescicole citometria a flusso

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Sincronizzazione di<em&gt; Caulobacter crescentus</em&gt; Per Indagine del ciclo cellulare batterica

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Thermo flusso LED - La combinazione Optogenetics con citometria a flusso

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Determinazione del relativo cellulari di superficie e di espressione totale di canali ionici ricombinante in citometria a flusso

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combinazione di sincronizzazione cellulare mitotica e microscopia confocale ad alta risoluzione per studiare il ruolo delle proteine del ciclo cellulare multifunzione durante la mitosi

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...