Name: preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)
Uploaded: 2012-06-18T13:00:00
Description: 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

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</sup...

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

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