We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).

1. FUCCI trasduzione e colture cellulari <ol><li> FUCCI trasduzione <ol><li> Creare linee cellulari che esprimono stabilmente la FUCCI costruisce mKO2-hCdt1 (30-120) e MAG-hGem (1-100) 15 usando lentivirus co-trasduzione come descritto in precedenza 7. Nota: il sistema FUCCI è ora disponibile in commercio. </li><li> Generare sub-cloni con fluorescenza mediante selezione singola cellula. Ordina cellule singole positive sia per AG e KO (giallo) per la fluorescenza delle cellule attivate l&#39;ordinamento in una piastra a 96 pozzetti come in precedenza descritto 7,16. </li></ol></li><li> Coltura cellulare Melanoma <ol><li> Cellule di melanoma umano Cultura C8161 come precedentemente descritto 7,17. </li></ol></li></ol> 2. 3D Spheroid Formazione (come descritto in precedenza 3,9) <ol><li> Piatto preparazione Agarosio <ol><li> Sciogliere 1,5% agarosio (o nobile agar) in 100 ml di soluzione di Hank salina bilanciata (HBSS) o Buffere fosfatod (PBS), facendo bollire nel microonde per 3-5 minuti, rimestando. Assicurarsi che l&#39;agarosio sia completamente dissolto. </li><li> Immediatamente dispensare 100 microlitri della soluzione di agarosio per bene ad una piastra a 96 pozzetti di coltura tissutale a fondo piatto con una pipetta multicanale. Nota: Agarosio può solidificare nelle punte dopo un breve periodo di tempo, per superare questo consigli problema deve essere cambiato tra piastre se effettuare più di una piastra di agarosio. </li><li> Lasciare piastra a 96 pozzetti su una superficie piana per indurire agarosio per almeno 1 ora. </li></ol></li><li> La preparazione delle cellule e di sovrapposizione su agarosio <ol><li> Crescere le cellule a circa l&#39;80% di confluenza in un pallone T75. Lavare con 10 ml di HBSS. </li><li> Staccare le cellule con 1,5 ml di 0,05% tripsina / acido etilendiamminotetraacetico (EDTA) e risospendere in 10 ml di terreno normale. </li><li> Contare le cellule utilizzando un emocitometro 18 o un altro metodo di conteggio automatizzato. </li><li> Risospendere le cellule a una concentrazione finale di 25.000 cellule / ml a normaal medio. </li><li> Overlay pozzi agarosio con 200 microlitri della sospensione cellulare per un numero finale di 5000 cellule per pozzetto. </li><li> Piastre ritorno alla incubatore di coltura cellulare (5% di CO 2 e 37 ° C) per formare sferoidi oltre circa 3 giorni. Nota: Il tempo impiegato per formare uno sferoide varia tra diverse linee cellulari. Alcune linee cellulari non formano sferoidi utilizzando questo metodo a tutti. </li><li> Immagine sferoide morfologia con un microscopio invertito con obiettivo 4X e un filtro a contrasto di fase. Una linea di cellule che si forma con successo sferoidi produrranno compatti, aggregati di cellule più o meno sferiche, e non ci dovrebbero essere solo uno sferoide per pozzetto. Nota: Opzionale: Una volta sferoidi si sono formate, possono essere trapiantate in collagene o altro matrice per ulteriori analisi di crescita e l&#39;invasione 3,7,17. Per rimuovere sferoidi da collagene, trattare con 500 ml di 2 mg / ml di collagenasi a 37 ° C fino a quando il collagene è sciolto abbastanza per il spherOID di entrare gratis nei media (circa 30 minuti). Rimuovere delicatamente sferoidi con una pipetta 1 ml. </li><li> Per il sezionamento / immagini sferoidi fix (o isolati da collagene o coltivate per 3-5 giorni in agarosio) nel 10% formalina tamponata neutra (ATTENZIONE: formalina deve essere usato sotto cappa) per almeno 2 ore a temperatura ambiente (RT) , o per una notte (O / N) a 4 ° C. Sferoidi possono essere conservati in HBSS a 4 ° C per diversi giorni. </li></ol></li></ol> 3. Spheroid Vibratome sezionamento <ol><li> Sciogliere 5 g di agarosio a basso punto di fusione in 100 ml di HBSS in una bottiglia di vetro da 500 ml in un forno a microonde. Utilizzare un calore medio, rimestando. ATTENZIONE: Tenere il coperchio sciolto quando bolle la soluzione in modo che la pressione può essere rilasciata. Usare guanti protettivi resistenti al calore per proteggere le mani da ustioni quando si maneggia la bottiglia di vetro caldo. </li><li> Attendere che l&#39;agarosio si raffredda un po &#39;- quindi la bottiglia può essere toccato senza bruciare le mani. </li><li> Pour approssiTely 2 ml di agarosio nella parte inferiore di un contatore Coulter tazza o simile plastica stampo. Aspirare immediatamente un sferoide fisso (vedi paragrafo 2.2.8) con una pipetta 1 ml nel più piccolo volume di liquido possibile. Aggiungere sferoide alla agarosio. Alcuni sferoidi (fino a 10) possono essere aggiunti per tazza. </li><li> Coprire i sferoidi con 2 ml più agarosio. Fare questo rapidamente per evitare l&#39;indurimento agarosio prima di aggiungere il secondo strato. </li><li> Lasciare che il agarosio indurire a temperatura ambiente al buio per almeno 3 ore. A questo punto tazze possono essere conservati per un breve periodo a 4 ° C con 2-3 ml di HBSS sovrapposti per evitare la disidratazione. </li><li> Rimuovere agarosio dalla tazza. Tagliare l&#39;agarosio contenente sferoidi modo che si inserisca sul blocco di metallo vibratome, e sferoidi sono vicino alla parte superiore della agarosio. Sferoidi possono essere visti come piccoli oggetti bianchi dentro il agarosio. Super Glue l&#39;agarosio al blocco. </li><li> Riempire il vibratome con acqua distillata e montare il blocco nel vibratome. Impostare ilvibratome per tagliare 100 micron sezioni utilizzando bassa velocità (impostazione 2) e ad alta vibrazione (impostazione 8). Impostare l&#39;angolo lama di taglio a 16 °. Accendere vibratome su, accendere la luce vibratomo e tagliare sezioni dalla cima della agarosio fino a 100 micron sezioni sferoidali sono tagliati. Posizionare tagliate sezioni sferoidali in HBSS in una piastra da 24 pozzetti. </li><li> Scegli sezioni centrali per l&#39;analisi delle immagini. Montare sezioni su vetrini mediante il trasferimento della sezione piatta sul vetrino con una pinzetta. Riempire una siringa da 10 ml con grasso per vuoto; aggiungere una linea sottile di grasso per vuoto intorno ai bordi della sezione per evitare che il supporto di montaggio di scappare bordi della diapositiva e contribuire a sigillare la sezione sotto il coprioggetto. Coprire la sezione con mezzo di montaggio e un vetrino. Sigillare i bordi del coprioggetto con smalto. Conservare scivola a 4 ° C prima di imaging. </li></ol> 4. confocale Image Acquisition <ol><li> Prendete una immagine z fetta di una sezione centrale sferoide FUCCI utilizzando un 10Xo obiettivo 20X come descritto in precedenza 7. Assicurarsi che non si sovrappongono o diafonia si verifica tra il Kusabira Orange2 e Azami Verde. Se si confrontano analisi delle immagini tra più sezioni sferoidali, mantenere potenza del laser e altre impostazioni dello stesso tra i campioni. </li></ol> 5. Hoechst Dye Diffusion Assay for Flow Ordinamento <ol><li> Trasferire sferoidi vivi (3-5 giorni dopo la semina agarosio) ad un tubo da 15 ml. Sia sferoidi gravità si depositano sul fondo della provetta. Diversi sferoidi possono essere colorati per provetta. Circa 20 sferoidi sono necessari per ottenere il numero di cellule sufficiente per citometria a flusso. </li><li> Rimuovere eccesso di terreno e aggiungere 1 ml di 10 micron Hoechst diluiti in mezzo normale. Incubare sferoidi a 37 ° C per circa 1 ora. Utilizzare dolce rapido passaggio per risospendere sferoidi una o due volte durante l&#39;incubazione. Nota: Il tempo di incubazione può essere variata di modificare fino a che punto il colorante Hoechst penetra nei sferoidi. Questo varia in basela dimensione sferoide, densità e linea cellulare. Per alcuni grandi / sferoidi densi ci può essere una massima penetrazione colorante che è inferiore al 100%. </li><li> Lavare i sferoidi delicatamente con 5 ml di HBSS con gravità sedimentazione. <ol><li> Opzionale: Per l&#39;imaging di penetrazione del colorante: Fix sferoidi con il 10% formalina tamponata neutra per almeno 2 ore a temperatura ambiente o O / N a 4 ° C. Preparare vibratome sezioni, montare sulle diapositive e immagini sul confocale come descritto ai punti 3 e 4 di cui sopra. </li></ol></li><li> Per preparare le cellule per citometria a flusso, trypsinize sferoidi con 1 ml di 0,05% tripsina / EDTA a 37 ° C per circa 30 minuti con sfogliando ogni 10 min per rompere sferoidi parte. Nota: Spheroids sono difficili da ottenere in sospensione singola cella, le condizioni possono avere bisogno di essere ottimizzato. Viabilità di C8161 4 giorni di età sferoidi dopo questo processo è di circa il 95%. </li><li> Cellule macchia con un live nel vicino infrarosso / macchia morti secondo il protocollo produttori. Lavare con HBSS quindi fissare con 1ml di 10% formalina tamponata neutra per circa 30 min. Le cellule possono essere conservate in HBSS a 4 ° C per diversi giorni prima analisi del flusso. </li></ol> 6. Citometria a flusso Analisi di Hoechst Stained Fucci Spheroids <ol><li> Assicurarsi che le cellule non sono raggruppate insieme facendoli passare attraverso un colino cella. Risospendere il Hoechst macchiato sferoidi Fucci in ghiaccio freddo FACS lavaggio (PBS con 5% Fetal Calf Serum) ad una concentrazione di 1-5 x 10 6 cellule / ml. Trasferire 200 ml di campione in 5 ml provette rotonde di fondo. </li><li> Preparare i seguenti singoli campioni di controllo del colore, come indicato al paragrafo 6.1: cellule di melanoma non-macchiato; cellule di melanoma aderenti colorate con 10 mM Hoechst per 1 ora; Azami verde solo esprimendo cellule di melanoma; e Kusabira arancione solo esprimendo cellule di melanoma. Nota: controlli monocromatici possono essere fissati in formalina e conservati in FACS lavare con 0,1% sodio azide a 4 ° C per settimane o mesi. </li><li> Sospensioni di cellule singole o Runna citometria a flusso analizzatore con i laser appropriati e singolo colore / controlli non colorati come descritto in precedenza 7,16. </li><li> Utilizzare il software citometria commerciale come FlowJo per analizzare l&#39;interno contro cellule sferoidali esterne come segue: <ol><li> Rimuovere i detriti dal gating sulla popolazione di cellule principale in Light Forward Scattered (FSC) vs. Luce sparsi laterale (SSC). </li><li> Rimuovere doppiette da gating su singole cellule utilizzando FSC (Regione) vs. FSC (altezza) </li><li> Porta per le cellule dal vivo di gating sulla popolazione dal vivo / morto basso. </li><li> Definire Hoechst cellule di alta gated, in base al segnale positivo dalle cellule aderenti colorate con Hoechst, come cellule &quot;esterni&quot;. </li><li> Definire Hoechst cellule bassi o negativi, gated sulla base del segnale dal controllo non-tinto, come cellule &quot;interni&quot;. </li><li> Dopo gating per le popolazioni interne ed esterne, le cellule possono essere ulteriormente gated per FUCCI rosso (G1), giallo (inizio S), verde (S / G2 / M) o negativo (G1 precoce). Nota: Cn aiuto compensativo utilizzando i comandi a singolo colore può essere richiesto di definire in modo adeguato le popolazioni rosso, giallo, verde e negativi Fucci. </li></ol></li><li> Una volta che il test è stato ottimizzato e la penetrazione Hoechst convalidato tramite microscopia confocale, eseguire cellule senza fissaggio su un cell sorter di flusso come descritto in precedenza 7,16 per ulteriori analisi di cellule vive sotto-popolazioni. </li></ol> Analisi 7. Immagine di FUCCI sferoide Sezioni <ol><li> Aprire il software ad es. Volocity, scegliere l&#39;unica configurazione quantificazione. </li><li> Creare una nuova libreria. Importare il FUCCI sferoide file immagine confocale nel software trascinando il file di dati grezzi dal confocale nella libreria. In alternativa, utilizzare il comando File&gt; Importa. </li><li> Vai alla scheda misure per costruire un protocollo di analisi delle immagini. Il protocollo è costruito trascinando i comandi appropriati nell&#39;ordine corretto come segue nella finestra del protocollo. </li><li> Trova objette nel canale verde: Trascinare il comando (che si trova in &#39;Finding&#39;) nella finestra del protocollo il ritrovamento di oggetti, selezionare il canale corretto negli oggetti find protocol.Add un comando di apertura (che si trova in &#39;Processing&#39;), poi un toccare oggetti separati il comando (che si trova in &#39;trattamento&#39; - questo sarà separare le cellule). Escludere gli oggetti di dimensioni &lt;50 micron (che si trova in &#39;filtraggio&#39; - questo esclude piccoli oggetti non cellulari). Nota: Le variabili quali oggetti il ​​ritrovamento di soglia, il numero di iterazioni aperte, le dimensioni degli oggetti da separare e le dimensioni degli oggetti da escludere deve essere ottimizzata manualmente fino maschere oggetto corrispondenza dell&#39;immagine. </li><li> Trova gli oggetti nel canale rosso: Ripetere trovare oggetti come sopra per il canale rosso. Potrebbe essere necessario modificare separatamente protocolli verde e rosso. Nota: A causa di nuclei essere in G2, nuclei verdi sono spesso leggermente più grandi. </li><li> Confermare oggetti ritrovati sono accurate Confermare rosso e oggetti verdi corrispondono allanuclei rosso e verde nell&#39;immagine spegnendo e sui canali verde e rosso (con la pelle o mostrare comando canale) durante la visualizzazione delle maschere rosso e verde, trovate da protocollo. Le opzioni di retroazione (Misurazioni&gt; opzioni di retroazione) possono essere utilizzati per modificare l&#39;aspetto delle maschere oggetto. </li><li> Trova gli oggetti gialli (cellule in fase precoce S): Per trovare celle gialle, aggiungere un intersecano rosso con le cellule verdi di comando (si trovano in &#39;combinazione&#39;). Escludere gli oggetti per dimensione (&lt;50 micron, si trovano in &#39;filtraggio&#39;) di escludere i piccoli oggetti non cellulari. </li><li> Trova gli oggetti esclusivamente rossi (cellule in fase G1): Per trovare i nuclei esclusivamente rossi, sottrarre le cellule gialle dai globuli rossi (che si trova in &#39;combinazione&#39;). Escludere gli oggetti in base alle dimensioni &lt;50 micron, (che si trova in &#39;filtraggio&#39;) per eliminare eventuali piccoli oggetti non cellulari creati. </li><li> Trova gli oggetti esclusivamente verdi (S / G2 / M celle di fase): Ripetere la procedura per il canale verde di trovare esclusivamente verde. Note: Se ci sono ancora oggetti non cellulari restanti, un comando popolazione filtro può essere aggiunta ai protocolli di rosso verde o esclusive Exclusive (che si trova in &#39;filtraggio&#39;) di mantenere solo gli oggetti con un fattore di forma maggiore di 0,25. Questo eliminerà gli oggetti non circolari. </li><li> Trova il contorno sferoide: Trova il contorno sferoide utilizzando un ritrovamento di comando (che si trova in &#39;Finding&#39;) con il canale verde o rosso (a seconda di quale ha più celle intorno al bordo dello sferoide) oggetti. Una soglia molto più bassa (che si trova nel trovare gli oggetti variabili) dovrà essere usato per trovare il contorno sferoide rispetto alle singole celle. <ol><li> Utilizzare un comando di chiusura per unire gli oggetti (che si trova in &#39;Processing&#39;), poi riempire i fori negli oggetti (che si trova in &#39;Processing&#39;), quindi utilizzare un filtro fine (che si trova in &#39;filtraggio&#39;) per rimuovere il rumore da oggetti. Infine, escludere gli oggetti in base alle dimensioni per rimuovere gli oggetti più piccoli &lt;30.000 micron (a seconda delle dimensioni sferoide). Nota: Questo protocollo dovrà essere ottimizzato manualmente finché il contorno sferoide è accurato. Un&#39;immagine brightfield può essere utilizzato anche - ma questo è generalmente meno accurato con una sezione sferoide, e un comando di inversione (trovato in &#39;Combinando&#39;) dovrà essere utilizzato. </li></ol></li><li> Misurare le distanze di nuclei a contorno sferoide: Misurare le distanze (che si trova in &#39;Relative&#39;) dalle popolazioni di colore giallo, esclusivamente verde e rosso esclusivamente dal baricentro cella a bordo della sferoide outline.To visualizzare le distanze minime, che dovrebbe essere dal cella a bordo sferoide più vicino, attivare Mostra distanze nella scheda relazioni di opzioni di feedback (Misure&gt; Le opzioni di retroazione&gt; Relazioni). </li><li> Salvare il protocollo. Questo protocollo può essere ri-applicato ad altre immagini utilizzando le misurazioni&gt; Comando Restore protocollo. </li><li> Creare un elemento di misure (Misure&gt; Fai Misura articolo). I dati possono essere analizzati utilizzando l&#39;analisifunzioni. <ol><li> Passare alla scheda analisi della voce misurazioni. Vai al menu Analysis (Analisi&gt; Analisi) e analizzare la minima distanza dal baricentro delle cellule (rosso, verde o giallo) al bordo sferoide, riassunta dal conte e organizzato per popolazione. </li><li> Per contare il numero di cellule che si trovano ad una certa distanza dal bordo creare un filtro (Analisi&gt; Filter). Il filtro per la distanza minima in figura 2 si basa sulla penetrazione Hoechst (es distanza minima è inferiore a 80 dà popolazione &quot;esterna&quot;). In alternativa, esportare i dati grezzi di un foglio di calcolo per ulteriori analisi. </li></ol></li></ol>

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M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> Journal of visualized experiments : JoVE. , (2010).</li><li>Smalley, K. S., 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=Up-regulated+expression+of+zonula+occludens+protein-1+in+human+melanoma+associates+with+N-cadherin+and+contributes+to+invasion+and+adhesion.">Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion.</a> The American journal of pathology. 166, 1541-1554 (2005).</li><li>Wang, Q., 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=Targeting+glutamine+transport+to+suppress+melanoma+cell+growth.">Targeting glutamine transport to suppress melanoma cell growth.</a> International journal of cancer. Journal international du cancer. 135, 1060-1071 (2014).</li><li>Lorenzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+division+dynamics+monitoring+in+3D+large+spheroid+tumor+models+using+light+sheet+microscopy.">Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy.</a> Cell division. 6, 22 (2011).</li><li>Pampaloni, F., Ansari, N., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+deep+imaging+of+live+cellular+spheroids+with+light-sheet-based+fluorescence+microscopy.">High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy.</a> Cell and tissue research. 352, 161-177 (2013).</li><li>Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Hoechst+33342+for+cell+selection+from+multicell+systems.">Use of Hoechst 33342 for cell selection from multicell systems.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 30, 117-122 (1982).</li><li>Laurent, 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=Multicellular+tumor+spheroid+models+to+explore+cell+cycle+checkpoints+in+3D.">Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D.</a> BMC cancer. 13, 73 (2013).</li><li>LaRue, K. E., Khalil, M., Freyer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+proliferation+in+multicellular+spheroids+is+mediated+through+differential+expression+of+cyclin-dependent+kinase+inhibitors.">Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.</a> Cancer research. 64, 1621-1631 (2004).</li><li>Kyle, A. H., Huxham, L. A., Baker, J. H., Burston, H. E., Minchinton, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+distribution+of+bromodeoxyuridine-labeled+cells+is+strongly+dose+dependent.">Tumor distribution of bromodeoxyuridine-labeled cells is strongly dose dependent.</a> Cancer research. 63, 5707-5711 (2003).</li><li>Minchinton, A. I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nature reviews. Cancer. 6, 583-592 (2006).</li><li>Giesbrecht, J. L., Wilson, W. R., Hill, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiobiological+studies+of+cells+in+multicellular+spheroids+using+a+sequential+trypsinization+technique.">Radiobiological studies of cells in multicellular spheroids using a sequential trypsinization technique.</a> Radiation research. 86, 368-386 (1981).</li></ol>

PerkinElmer contributed to the costs of publication.

Analisi delle immagini semi-automatica ha identificato la G1 arrestato regione interna sferoide, e proliferare strati esterni. Questo metodo può essere utilizzato su sferoidi vivi utilizzando una sezione ottica, o in sezioni sferoidali fissi, per identificare variazioni non solo il ciclo cellulare ma espressione marcatore (via immunofluorescenza), morte cellulare, o morfologia cellulare in queste regioni diverse. Motilità cellulare in diverse regioni sferoidali può anche essere quantificato - se si aggiunge dal vivo ...

Sferoidi multicellulare 3D sono stati conosciuti come un modello di tumore per decenni, ma è solo di recente che sono venuti in uso più comune come un modello in vitro per molti tumori solidi. Essi sono sempre più utilizzati in high-throughput schermi scoperta di nuovi farmaci come intermedio tra complessi, costosi e in modelli in vivo che richiede tempo e il semplice, il modello 2D monostrato basso costo 1-6. Gli studi in coltura 2D sono spesso incapaci di replicare in vivo. Modelli sferoidali di molti tipi di cancro sono in grado di imitare le caratteristiche di crescita, la sensibilità ai farmaci, di penetrazione della droga, interazioni cellula-cellula, disponibilità limitata di ossigeno e nutrienti e lo sviluppo di necrosi che si vede in vivo nei tumori solidi 6-11. Spheroids sviluppano un nucleo necrotico, una regione arrestato quiescente o G1 che circonda il nucleo, e le cellule proliferanti alla periferia dello sferoide 7. Lo sviluppo di queste regionipuò variare a seconda della densità cellulare, tasso di proliferazione e la dimensione dello sferoide 12. E &#39;stato ipotizzato che l&#39;eterogeneità cellulare visto in queste diverse sotto-regioni può contribuire a cancro resistenza alla terapia 13,14. Pertanto, la capacità di analizzare le cellule in queste regioni è parte fondamentale per la comprensione risposta ai farmaci tumorali. Il sistema di indicatori del ciclo cellulare fluorescenza ubiquitination (FUCCI) si basa sul rosso (Kusabira Orange - KO) e verde (Azami verde - AG) il tagging fluorescente CDT1 e Geminin, che vengono degradati nelle diverse fasi del ciclo cellulare 15. Così nuclei delle cellule appaiono di colore rosso in G1, gialli in S e verde in S / G2 / M di fase. Descriviamo qui due metodi complementari sia con FUCCI per identificare il ciclo cellulare, insieme con l&#39;uso di software di imaging o di un flusso di colorante diffusione citometria test per determinare se le cellule risiedono nel centro G1 arrestato o proliferatin esternog anello, e la distanza delle singole cellule dal bordo dello sferoide. Questi metodi sono stati sviluppati nella nostra pubblicazione precedente, dove abbiamo dimostrato che le cellule di melanoma nelle regioni ipossiche nel centro dello sferoide e / o in presenza di terapie mirate sono in grado di rimanere in G1 arresto per lunghi periodi di tempo, e può ri- entrare nel ciclo cellulare in cui condizioni più favorevoli emergono 7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>agarose low melting point</td>
			<td>Life Technologies</td>
			<td>16520-050</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>noble agar&#160;</td>
			<td>Sigma</td>
			<td>A5431</td>
			<td>For making spheroids</td>
		</tr>
		<tr>
			<td>agarose for spheroids</td>
			<td>Fisher Scientific</td>
			<td>BP1356-100</td>
			<td>For making spheroids</td>
		</tr>
		<tr>
			<td>0.05% trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin</td>
			<td>Sigma</td>
			<td>HT5014-1CS</td>
			<td>CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do &#160;not breath fumes (open in a fume cupboard).</td>
		</tr>
		<tr>
			<td>live/dead near IR</td>
			<td>Life Technologies</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>vibratome</td>
			<td>Technical Products International, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>coulture cup</td>
			<td>Thermo-Fisher Scientific</td>
			<td>SIE936</td>
			<td>Mold for sectioning spheroids</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Invitro</td>
			<td>FAL353072</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Sigma</td>
			<td>C5138&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer analyser</td>
			<td>Becton Dickinson</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>flowjo</td>
			<td>Tree Star</td>
			<td></td>
			<td>Flow cytometry software</td>
		</tr>
		<tr>
			<td>Vaccuum grease</td>
			<td>Sigma</td>
			<td>Z273554</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FUCCI (commercial constructs)</td>
			<td>Life Technologies</td>
			<td>P36238</td>
			<td>Transient transfection only</td>
		</tr>
		<tr>
			<td>Cell strainer 70 um</td>
			<td>In Vitro</td>
			<td>FAL352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom 5 mL tubes (sterile)</td>
			<td>In Vitro</td>
			<td>FAL352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom 5 mL tubes (non-sterile)</td>
			<td>In Vitro</td>
			<td>FAL352008</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3d melanoma spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

Esistono diversi metodi per produrre sferoidi tumorali, questo protocollo utilizza il metodo di crescita non aderente, in cui le cellule sono coltivate su agar o 3,7,9 agarosio. La Figura 1 mostra un esempio di uno sferoide C8161 melanoma dopo 3 giorni su agar. Sferoidi C8161 formano sferoidi di dimensioni regolari con un diametro di 500 - 600 micron (media = 565, DS = 19, n = 3) dopo 3 giorni. Altre linee di cellule di melanoma che formeranno sferoidi includono: WM793, WM983C, WM983B, WM164,...

Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Analisi basata su Citometria Imaging- e di flusso della posizione cellulare e ciclo cellulare in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

imaging-flow-cytometry-based-analysis-cell-position-cell-cycle-3d

Research

JoVE Journal

Medicine

12.9K Views.  The Centenary Institute. We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids. 

Video: Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Ricerca

Medicina

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Lavorazione dei tessuti primaria tumore al cervello per saggi su cellule staminali e flusso di Ordinamento

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria a Flusso test cellulare per individuare gli anticorpi per N-metil-D-aspartato Receptor o dopamina-2 recettore nel siero umano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adattamento del semiautomatico circolanti (CTC) Saggi Tumor Cell per applicazioni cliniche e di ricerca preclinica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa Bengala Photothrombosis da confocale Optical Imaging<em&gt; In Vivo</em&gt;: Un modello di singolo vaso Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Un'analisi basata su citometria a flusso per misurare il potenziale di membrana mitocondriale in miociti cardiaci dopo ipossia/riossigenazione

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Analisi della citometria del flusso di sottoinsiemi di cellule immunitarie all'interno della milza murina, del midollo osseo, dei nodi linfociti e del tessuto sinoviale in un modello di osteoartrite

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.