Riconosciamo il Fondo flusso Cytometry USF Core. Vorremmo ringraziare il Dott. Denise Cooper per la condivisione di risorse. Vorremmo anche ringraziare Maya Cohen, Laura Pendleton e Diana Latour per la loro assistenza nella creazione e le riprese di questo video. NN supportato da NSF FG-LSAMP Ponte al Dottorato Fellowship HRD # 0929435. Questo lavoro è stato finanziato dalla American Society istituzionale Cancer Research Centre di Grant # 93-032-13/Moffitt Cancer assegnato al TG.

Prima di iniziare, preparare le seguenti soluzioni: 3% colorazione Media (SM): -3% Siero fetale bovino (FBS) in 1X tampone fosfato salino (PBS) MACS Buffer (MB): - 0,5% di albumina da siero bovino (BSA) in 1XPBS 1. Harvest milza di topi <ol><li> Iniettare per via sottocutanea 6-8 settimane di età C57BL / 6 topi (Harlan) con 1,5 x 10 5 cellule murine Panc02 sospesi in 100 microlitri di PBS 1x (portatore di tumore, TB). Topi di controllo (Naïve) ricevono 100 1XPBS pl. </li><li> Circa 4 settimane dopo l&#39;iniezione, i topi eutanasia per asfissia di anidride carbonica. </li><li> Harvest milza di topi per via smussa con pinze e forbici, quindi pesare con una bilancia. Luogo in separata milza, etichettato provette da 50 ml coniche contenenti 1 x PBS. </li></ol> 2. Generare una cella singola Suspension dei leucociti da milze Tutte le operazioni devono essere eseguite in un ambiente sterile sotto una cappa di protezione biologica e le cellule e gli anticorpi tenuti su ghiaccio. <ol><li> Montare la dissociazione cellulare setaccio inserendo lo schermo maglia nell&#39;apertura del bicchiere verso il basso. Quindi, inserire l &#39;anello nella zona filettata con il lato scanalato e utilizzare il key ring per stringere l&#39;anello di sostegno, tenendo così lo schermo in posizione. Posizionare il setaccio montato in una piastra Petri 10 ml contenente 1 x PBS. </li><li> Milza piscina e pestello in vetro uso per macinare milza contro lo schermo maglie della cellula setaccio dissociazione e nella piastra di Petri. Ripetere l&#39;operazione per ciascun gruppo di trattamento di topi. </li><li> Sospensione cellulare filtro in una provetta da 50 ml con un colino cella 70 micron e una pipetta da 5 ml sierologica. Centrifugare a 12.000 rpm (250-300 xg) per 5 min. </li><li> Rimuovere il surnatante e risospendere pellet in 5 ml 1 x tampone di lisi per RBC milza. Pipettare su e giù con forza. Incubare a temperatura ambiente per 5 min. Arrestare la reazione aggiungendo 20 ml di 1 x PBS. Pipettare su e giù con forza. Centrifugare a 12.000 rpm (250-300 xg) per 5 min. </li><li> Rimuovere il surnatante e risospendere pellet in 20 ml 1 x PBS sterile. Pipettare su e giù con forza. </li><li> Contare le cellule con tripan blu e un emocitometro e risospendere alla concentrazione desiderata in 3% SM in modo che 50 pl è equivalente a 5x10 5-1x10 6 cellule (1x10 7 cellule / ml - 2x10 7 cellule / ml). </li></ol> 3. Cell-superficie colorazione / Immunofenotipizzazione MDSC della citometria a flusso <ol><li> Etichettare i pozzetti di una piastra a 96 pozzetti fondo a V per il controllo e campioni sperimentali e macchie singole per i controlli di compensazione (No Stain, NS, Mac-1-FITC; Gr-1-APC e DAPI). </li><li> Aggiungere 5x10 5-1x10 6 cellule equivalenti a 50 pl / pozzetto di splenociti ai relativi pozzetti in 96-ben-V piastra inferiore. CentrifugaPiastra a 12.000 rpm (250-300 xg) per 5 min. </li><li> Preparare &quot;Mix Master&quot; (MM) di Mouse BD Fc Block (Rat anti-topo CD16/32 anticorpo monoclonale) diluito in 3% SM, in una provetta 1,5 ml su ghiaccio. Come punto di partenza, utilizzare 1 Block ug Fc in 3% SM per un volume finale di 50 pl, per pozzetto. </li><li> Rimuovere con cautela il surnatante da ogni pozzetto del 96 e V-bottom piastra invertendo rapidamente la piastra sopra e ritorno, su un contenitore per rifiuti o nel lavandino, senza interruzione dei pellet cellulari. </li><li> Vortex, centrifugare brevemente MM Block Fc per 5 secondi e aggiungere 50 microlitri a tutti i pellets in ben 96 V-piastra inferiore. Mescolare bene pipettando gentilmente su e giù, lasciando i campioni nei pozzetti. Incubare la piastra per 15 minuti al buio su ghiaccio. Centrifugare piastra a 12.000 rpm (250-300 xg) per 5 min. </li><li> Preparare &quot;Mix Master&quot; (MM) di anticorpi coniugati con fluorocromo diluito in 3% SM in una provetta 1,5 ml su ghiaccio, mentre i campioni incubare con blocco Fc. Gli anticorpi devono essere titolatodeterminare diluizioni ottimali per procedure di colorazione. Come punto di partenza, combinare una diluizione 1:25 di Mac-1-FITC e 1:20 diluizione di Gr-1-APC in 3% SM per un volume finale di 50 pl, per pozzetto. </li><li> Rimuovere attentamente surnatante da ciascun pozzetto della ben-96 V-basso come precedentemente descritto. </li><li> Vortex, centrifugare brevemente MM di fluorescenza anticorpi coniugati colorazione per 5 secondi e aggiungere 50 microlitri di controllo e pellets sperimentali. Mescolare bene pipettando gentilmente su e giù. Per singola macchia compensazioni, aggiungere una diluizione 1:25 di Mac-1-FITC, 1:20 diluizione di Gr-1-APC e 75 ng / ml di DAPI, 3% in SM per un volume finale di 50 pl per pozzetto a relativi pozzetti. Aggiungere 50 microlitri SM 3% senza macchia e (senza macchia). Mescolare bene e incubare cellule in ben 96 V-piastra inferiore per 30 minuti al buio in ghiaccio. </li><li> FACS Etichettare provette (5 ml, 12 mm x 75 mm provette di polistirene a fondo tondo) per corrispondere a ciascun pozzetto della piastra a 96 pozzetti V-basso. Aggiungere 200 ul SM 3% ad ogni FACStubo. </li><li> Centrifugare piastra a 12.000 rpm (250-300 xg) per 5 minuti e rimuovere supernatanti. Lavare pellet aggiungendo 100 microlitri SM 3% ad ogni pellet e mescolare bene pipettando gentilmente su e giù. Centrifugare piastra a 12.000 rpm (250-300 xg) per 5 min. Ripetere ancora una volta la fase di lavaggio. </li><li> Rimuovere attentamente surnatante da ciascun pozzetto della ben-96 V-basso come precedentemente descritto. Risospendere ogni pellet in 100 SM ul 3% e mescolare bene. </li><li> Trasferire 100 pl pellet risospeso da ciascun pozzetto della 96-ben-V piastra inferiore alla loro tubo rispettivamente etichettati FACS. </li><li> Prima analisi del flusso di citometria, aggiungere 75 ng / ml DAPI per il controllo e campioni sperimentali e di controllo DAPI singola macchia di compensazione. </li><li> Eseguire flusso di acquisizione di dati di citometria percentuali MDSC. Eseguire la compensazione utilizzando il negativo (nessun controllo macchia) e dei controlli singoli positivi. Impostare un dot plot che visualizza il forward scatter (FSC) rispetto a lato (SSC) in scala logaritmica, in modo che le popolazioni di leucocitiinteresse possono essere identificati. Disegnare un grande cancello su tutti i leucociti, ad eccezione detriti e macchie con il più basso in avanti e scatter laterale. Da questa porta principale, creare un nuovo grafico che visualizza dot SSC contro DAPI e cancello il DAPI-(live) delle cellule. Selezionare questa popolazione di recente gated e creare un dot plot che visualizza Mac-1 contro Gr-1 e porta sul doppio positive (Mac-1 + Gr-1 +) MDSC. </li></ol> 4. Arricchimento magnetica del GR-1 + Leucociti <ol><li> Aliquote 1x10 7 leucociti rimanenti non colorati in tubi opportunamente etichettati FACS e centrifugare a 12.000 rpm (250-300 xg) per 5 min. </li><li> Preparare MM di Gr-1-PE anticorpo in una provetta da 1,5 ml. Per un massimo di 10 7 celle, utilizzare una diluizione 1:10 del Gr-1-PE anticorpi in 50 MB pl, per campione. Per il numero di cellule più grandi, scalare i volumi di conseguenza. Brevemente centrifugare per 5 secondi, aggiungere a leucociti in tubi FACS e incubare per 15 min a 4 ° C al buio. </li&gt; <li> Aggiungere 2 ml di MB ai tubi FACS, centrifugare a 12.000 rpm (250-300 xg) per 5 minuti ed eliminare il surnatante. </li><li> Preparare MM di Anti-PE microsfere in una provetta 1,5 ml. Per un massimo di 10 7 celle, utilizzare una diluizione 1:04 di anti-PE microsfere in 200 MB pl, per campione. Per il numero di cellule più grandi, scalare i volumi di conseguenza. Brevemente centrifugare per 5 secondi, aggiungere a leucociti in tubi FACS e incubare per 15 min a 4 ° C al buio. </li><li> Aggiungere 2 ml di MB ai tubi FACS, centrifugare a 12.000 rpm (250-300 xg) per 5 minuti ed eliminare il surnatante. Risospendere il pellet in 3 ml di SB. Filtrare attraverso un colino 70 micron in un nuovo, etichettato tubo da 50 ml. </li><li> Preparare e prime Auto MACS Separator Pro. Refill tutte le bottiglie con le soluzioni più appropriate e svuotare la bottiglia dei rifiuti, se necessario. Accendere strumento ed esaminare lo stato dei contenitori di liquidi e di colonna (s) dopo l&#39;inizializzazione. Tutti i simboli dovrebbero essere di colore verde. Nel menu, selezionare &quot;separazione&quot; dal superiorebarra dei menu seguito da &quot;Wash Now&quot; dalla barra dei menu inferiore. Selezionare &quot;Risciacquare&quot; dal pop-up opzione seguito da &quot;Run&quot; per avviare il processo di adescamento. Una volta che il processo di adescamento è completata con successo, lo strumento visualizzerà quindi che si tratta di &quot;Ready for Separation&quot; sotto il menu Stato. </li><li> Scegli adatto a cremagliera refrigerate tubo per dimensioni dei tubi e il luogo tubo da 50 ml con le cellule marcate magneticamente nella riga A, tubo da 50 ml conica per la raccolta frazione negativa nella riga B e 15 ml tubo conico per la raccolta delle frazioni positive in fila C. </li><li> Scegliere il programma di separazione &quot;POSSEL_S&quot; cellula per la selezione positiva delle cellule bersaglio etichettati in modo sensibile dai campioni. Cellule bersaglio marcate magneticamente sono trattenuti sulla colonna tavole; cellule non marcate vengono rilasciati nel tubo di raccolta delle frazioni negativo in fila B. In retrazione automatica del magnete, le cellule bersaglio marcate verrà rilasciato nel tubo di raccolta positiva in fila C del tubo rack. </li></ol>+ Leucociti arricchite <ol><li> Recount Gr-1 + e Gr-1 - frazioni utilizzando trypan blu e un emocitometro. Risospendere le cellule a concentrazione desiderata nel mezzo colorazione 3% in modo che 50 pl è equivalente a 5x10 5-1x10 6 cellule (1x10 7 cellule / ml - 2x10 7 cellule / ml) e 50 pl per trasferire corrispondentemente etichettati tubi FACS. </li><li> Preparare una MM di Mac-1 solo, a una diluizione 1:25 a 3% SM per un volume finale di 50 pl per campione. Colorare le cellule e preparare singole compensazioni macchia di Gr-PE, Mac-1 FITC e DAPI per l&#39;analisi di citometria a flusso. Aggiungere 200 pl SM 3% e colorante vitalità DAPI come precedentemente descritto. </li><li> Eseguire la citometria a flusso di acquisizione dati per determinare Gr-1 + e Gr-1 - percentuali e di confrontare anche le percentuali di arricchimento MDSC pre-e post-autoMACS. </li></ol> 6. Risultati rappresentativi Qui vi mostriamo r<html> Risultati epresentative per l&#39;arricchimento autoMACS di Gr-1 + leucociti dal pool, milza naïve per FACS successive di smistamento di MDSC (Figura 1). 1x10 6 leucociti naive sono state colorate con Mac-1-FITC e Gr-1-APC anticorpi per identificare percentuali MDSC con uno strumento BD LSRII, prima di smistamento autoMACS. 1x10 7 leucociti naive sono state poi colorate con anticorpi anti-Gr-1-PE PE-anticorpi e microsfere per l&#39;arricchimento del Gr-1 + leucociti utilizzando un separatore autoMACS Pro. Post-autoMACS arricchimento, Gr-1 percentuali in Gr-1 + e Gr-1 - frazioni raccolte sono state valutate mediante citometria a flusso. 1x10 6 Gr-1 + leucociti sono stati rimossi e colorati con Mac-1-FITC anticorpi per analizzare e confrontare le percentuali di MDSC arricchito, naive agli aggregati leucociti non arricchito, pool portatori di tumore leucociti mediante citometria a flusso. fig1.jpg &quot;/&gt; Figura 1. Arricchimento AutoMACS di ingenuo Gr-1 + leucociti per FACS MDSC Ordinamento. Milze sono state raccolte dai topi portatori di tumore del pancreas e ingenuo e trasformati in unicellulari sospensioni. L&#39;analisi di citometria a flusso superficiale ingenuo leucociti colorati con Mac-1 e GR-1 fluorescente anticorpi coniugati, prima di arricchimento autoMACS (A). Analisi di citometria a flusso di Gr-1 + (B) e GR-1 - (C) frazioni post-autoMACS arricchimento di Gr-1 + cellule provenienti da un pool leuckocytes naïve colorati con Gr-1-PE e anticorpi anti-PE microsfere. Analisi di citometria a flusso di MDSC e Gr-1 + percentuali di post-autoMACS arricchimento di Gr-1 + cellule provenienti da un pool leucociti naïve (D) rispetto ai non-arricchiti leucociti raccolti da topi affetti da tumore (E) (topi naïve, n = 5 ; topi portatori di tumore, n = 3). MDSC e GR-1 percentuali sono gated nei grafici di contorno rappresentative e istogrammi.

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Questo è un metodo dettagliato per l&#39;elaborazione e la immunophentyping popolazioni MDSC che è applicabile a diversi tessuti linfoidi provenienti da vari modelli animali. In particolare, l&#39;arricchimento autoMACS può essere usato per l&#39;isolamento delle varie popolazioni di leucociti cui Gr-1 deplezione di splenociti 4, purificazione di sottoinsiemi mieloidi da splenociti e linfonodi 5, l&#39;isolamento dei neutrofili midollo osseo 14 e la purificazione di cellule T CD8 <sup...

<table border="1"><tbody><tr><td> REATTIVO </td><td> AZIENDA </td><td> Catalogo # </td><td> COMMENTI </td></tr><tr><td> 1X Phosphate Buffered Saline </td><td> Thermo Scientific Hyclone </td><td> SH30028.02 </td><td> Ca 2 + / 2 + Mg / rosso fenolo-free </td></tr><tr><td> Albumina da siero bovino (BSA) </td><td> Sigma-Aldrich </td><td> A7906 </td><td> Lasciate sciogliere indisturbati BSA in PBS; ambiente sterile </td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Thermo Scientific Hyclone </td><td> SV3001403HI </td><td> Inattivato calore; ambiente sterile </td></tr><tr><td> Rat anti-topo CD16/32 anticorpo monoclonale (Fc Block) </td><td> BD Biosciences </td><td> 553142 </td><td> Ambiente sterile </td></tr><tr><td> Anti-mouse CD11b (Mac-1) FITC </td><td> eBiosciences </td><td> 11-0112 </td><td> SterileAmbiente </td></tr><tr><td> Anti-mouse Ly6G (Gr-1) APC </td><td> eBiosciences </td><td> 17-5931 </td><td> Ambiente sterile </td></tr><tr><td> Anti-mouse Ly6G (Gr-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> Ambiente sterile </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> Diluizione Ambiente Serial Sterile </td></tr><tr><td> Dissociazione cellulare Sieve </td><td> Sigma-Aldrich </td><td> CD1-1KT </td><td> Autoclave prima dell&#39;uso </td></tr><tr><td> 70-um filtro </td><td> BD Biosciences </td><td> 352350 </td><td> Ambiente sterile </td></tr><tr><td> 1X RBC Lysis Buffer </td><td> eBiosciences </td><td> 00-4333-57 </td><td> Calda a temperatura ambiente prima dell&#39;uso; ambiente sterile </td></tr><tr><td> Scatole Petri </td><td> Fisher Scientific </td><td> 08-757-12 </td><td> Ambiente sterile </td></tr><tr><td> 50ml coniCal tubi </td><td> Thermo Scientific </td><td> 339652 </td><td> Ambiente sterile </td></tr><tr><td> 5ml 12X75mm polistirene con fondo arrotondato tubi </td><td> BD Biosciences </td><td> 352054 </td><td> Conosciuto come tubi FACS; ambiente sterile </td></tr><tr><td> 96 pozzetti V-piastre di fondo </td><td> Corning </td><td> 3897 </td><td> Ambiente sterile </td></tr><tr><td> Trypan blu </td><td> Cellgro </td><td> 25-900-CI </td><td> Ambiente sterile </td></tr><tr><td> PE microsfere </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> Ambiente sterile </td></tr><tr><td> AutoMACS Pro Separator </td><td> Miltenyi Biotec </td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS Colonne </td><td> Miltenyi Biotec </td><td> 130-021-101 </td><td></td></tr><tr><td> AutoMACS tampone di corsa </td><td> Miltenyi Biotec </td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

MDSC sono una popolazione eterogenea di macrofagi immature, cellule dendritiche e granulociti che si accumulano negli organi linfoidi in condizioni patologiche tra cui infezioni parassitarie, infiammazioni, stress traumatico, graft-versus-host disease, il diabete e il cancro 1-7. Nei topi, MDSC express Mac-1 (CD11b) e GR-1 (Ly6G e Ly6C) antigeni di superficie 7. È importante notare che MDSC sono state ampiamente studiate in vari portatori di tumore host dove vengono notevolmente aumentato e sopprimere le risposte immunitarie anti-tumorali rispetto alle controparti naive 7-10. Tuttavia, a seconda della condizione patologica, vi sono differenti sottopopolazioni di MDSC con meccanismi distinti e gli obiettivi di soppressione 11,12. Pertanto, i metodi efficaci per isolare vitali popolazioni MDSC sono importanti nel chiarire loro diversi meccanismi molecolari di soppressione in vitro e in vivo. Recentemente, il Ghansah gruppo ha riportato l&#39;espansione di MDSC in un modello murino cancro pancreatico. Il nostro portatore di tumore MDSC visualizzare una perdita di omeostasi e di aumento della funzione soppressiva rispetto al ingenua MDSC 13. MDSC percentuali sono significativamente meno in compartimenti linfoidi di topi naive contro tumore-cuscinetto. Si tratta di un avvertimento importante, che ostacola spesso accurate analisi comparativa di questi MDSC. Pertanto, arricchendo Gr-1 + leucociti dai topi naive prima Fluorescence activated cell sorting (FACS) esalta la purezza, la vitalità e riduce significativamente il tempo di ordinamento. Tuttavia, l&#39;arricchimento del Gr-1 + leucociti da topi affetti da tumore è facoltativo in quanto questi sono in abbondanza per FACS veloci ordinamento. Pertanto, in questo protocollo, si descrive un metodo molto efficiente di immunofenotipizzazione MDSC e arricchire Gr-1 + leucociti da milze di topi naive per l&#39;ordinamento MDSC in modo tempestivo. Immunocompetenti topi C57BL / 6 vengono inoculate con murino Panc02 ceLLS per via sottocutanea mentre i topi naïve ricevere 1XPBS. Circa 30 giorni dopo l&#39;inoculazione, milza vengono raccolti e trasformati in cella singola sospensioni utilizzando una cella di dissociazione setaccio. Splenociti sono poi globuli rossi (RBC) lisati e un&#39;aliquota di questi leucociti sono testate in base fluorocromo-coniugati anticorpi contro Mac-1 e Gr-1 a percentuali immunofenotipo MDSC citometria a flusso. In un esperimento parallelo, leucociti interi da topi naive sono macchiati coniugate con quelle fluorescenti-Gr-1 incubate con anticorpi, PE-microsfere e positivamente selezionato utilizzando un sistema automatizzato di smistamento magnetico cellulare Attivato (autoMACS) Pro Separator. Successivamente, una aliquota di GR-1 + leucociti vengono colorate con anticorpi Mac-1 per identificare l&#39;aumento percentuale MDSC citometria a flusso. Ora, queste GR1 + leucociti arricchiti sono pronti per FACS cernita di MDSC da utilizzare in analisi comparative (naïve vs portatore di tumore) in vivo e in vitro </em&gt; saggi.

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Questo è un metodo rapido e completo di immunofenotipizzazione cellule derivate mieloidi soppressore (MDSC) e arricchimento Gr-1<sup&gt; +</sup&gt; Leucociti dal milza del mouse. Questo metodo utilizza la citometria a flusso e le celle a AutoMACS Ordinamento per arricchire di vitale Gr-1<sup&gt; +</sup&gt; Leucociti prima FACS smistamento di MDSC per uso<em&gt; In vivo</em&gt; E<em&gt; In vitro</em&gt; Saggi.

Preparazione di cellule derivate mieloidi soppressore (MDSC) da Naive e Pancreatiche topi affetti da tumore in citometria a flusso e di smistamento automatico magnetico cellulare Attivato (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

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Research

JoVE Journal

Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

26.1K Views.  University of South Florida Morsani College of Medicine. Questo è un metodo rapido e completo di immunofenotipizzazione cellule derivate mieloidi soppressore (MDSC) e arricchimento Gr-1 + Leucociti dal milza del mouse. Questo metodo utilizza la citometria a flusso e le celle a AutoMACS Ordinamento per arricchire di vitale Gr-1 + Leucociti prima FACS smistamento di MDSC per uso In vivo E In vitro Saggi.

Video: Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

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 ...

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Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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 ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validazione di Nanobody e anticorpi Based<em&gt; In Vivo</em&gt; Esperimenti tumore xenotrapianto NIRF imaging nei topi mediante<em&gt; Ex Vivo</em&gt; Citometria di flusso e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Molecular Probe Optimization per determinare mortalità delle cellule in un organismo fotosintetico (<em&gt; Microcystis aeruginosa</em&gt;) In citometria a flusso

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

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.

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.