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

Vi anerkender USF Flowcytometri Core Facility. Vi vil gerne takke Dr. Denise Cooper til deling af ressourcer. Vi vil også gerne takke Maya Cohen, Laura Pendleton og Diana Latour for deres bistand til at oprette og filme i denne video. NN støttet af NSF FG-LSAMP Bridge til Doktorgrad Fellowship HRD # 0929435. Dette arbejde blev finansieret af American Cancer Society Institutional Research Grant # 93-032-13/Moffitt Cancer Center tildelt TG.

Før start, forberede følgende løsninger: 3% Farvning Media (SM): -3% Føtalt bovint serum (FBS) i 1X phosphatpuffersaltopløsning (PBS) MACS Buffer (MB): - 0,5% albumin fra bovint serum (BSA) i 1X PBS 1. Høst Milte fra mus <ol><li> Subkutant injektion 6-8 uger alder C57BL / 6 mus (Harlan) med 1,5 x 10 5 murine Panc02 celler suspen...

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Dette er en detaljeret metode til behandling og immunophentyping MDSC befolkninger, der gælder for forskellige lymfoide væv fra forskellige dyremodeller. Især kan autoMACS berigelse kan anvendes til isolering af forskellige leukocytpopulationer, herunder Gr-1 udtømning af splenocytter 4, oprensning af myeloide delmængder af splenocytter og lymfeknuder 5, isolering af knoglemarv neutrofiler 14 og oprensning af CD8 + T-celler fra milt og lymfeknuder 15. Uanset ce...

<table border="1"><tbody><tr><td> REAGENS </td><td> SELSKABET </td><td> Katalog # </td><td> KOMMENTARER </td></tr><tr><td> 1X phosphatpufret saltopløsning </td><td> Thermo Scientific Hyclone </td><td> SH30028.02 </td><td> Ca2 + / Mg2 + / phenolrødt-frit </td></tr><tr><td> Albumin fra bovint serum (BSA) </td><td> Sigma-Aldrich </td><td> A7906 </td><td> Lad BSA opløses uforstyrret i PBS; sterile omgivelser </td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> Thermo Scientific Hyclone </td><td> SV3001403HI </td><td> Varmeinaktiveret; sterilt miljø </td></tr><tr><td> Rotte antimus CD16/32 monoklonalt antistof (Fc Block) </td><td> BD Biosciences </td><td> 553142 </td><td> Sterile miljø </td></tr><tr><td> Anti-muse-CD11b (Mac-1) FITC </td><td> eBiosciences </td><td> 11-0112 </td><td> SterilMiljø </td></tr><tr><td> Anti-mus Ly6G (Gr-1) APC </td><td> eBiosciences </td><td> 17-5931 </td><td> Sterile miljø </td></tr><tr><td> Anti-mus Ly6G (Gr-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> Sterile miljø </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> Seriefortynding sterilt miljø </td></tr><tr><td> Celledissociering Sieve </td><td> Sigma-Aldrich </td><td> CD1-1KT </td><td> Autoklave før brug </td></tr><tr><td> 70 um si </td><td> BD Biosciences </td><td> 352350 </td><td> Sterile miljø </td></tr><tr><td> 1X RBC Lysis Buffer </td><td> eBiosciences </td><td> 00-4333-57 </td><td> Opvarmes til stuetemperatur før anvendelse; sterilt miljø </td></tr><tr><td> Petriskåle </td><td> Fisher Scientific </td><td> 08-757-12 </td><td> Sterile miljø </td></tr><tr><td> 50ml Conriske rør </td><td> Thermo Scientific </td><td> 339652 </td><td> Sterile miljø </td></tr><tr><td> 5 ml 12X75mm polystyren rundbundede rør </td><td> BD Biosciences </td><td> 352054 </td><td> Kendt som FACS-rør; sterilt miljø </td></tr><tr><td> 96-brønds V-bundformede plader </td><td> Corning </td><td> 3897 </td><td> Sterile miljø </td></tr><tr><td> Trypanblåt </td><td> Cellgro </td><td> 25-900-CI </td><td> Sterile miljø </td></tr><tr><td> PE Mikroperler </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> Sterile miljø </td></tr><tr><td> AutoMACS Pro Separator </td><td> Miltenyi Biotec </td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS Kolonner </td><td> Miltenyi Biotec </td><td> 130-021-101 </td><td></td></tr><tr><td> AutoMACS Running Buffer </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 er en heterogen population af umodne makrofager, dendritiske celler og granulocytter, der ophobes i lymfoide organer i patologiske tilstande, herunder parasitisk infektion, inflammation, traumatisk stress, graft-versus-host-sygdom, diabetes og cancer 1-7. I mus. MDSC udtrykker Mac-1 (CD11b) og Gr-1 (Ly6G og Ly6C) overfladeantigener 7 Det er vigtigt at bemærke, at MDSC godt undersøgt i forskellige tumor-bærende vært, hvor de er betydeligt udvidet og undertrykker anti-tumor immunrespons i forhold til naive modstykker 7-10. Afhængigt af den patologiske tilstand, der er forskellige subpopulationer af MDSC med forskellige mekanismer og mål suppression 11,12. Derfor effektive metoder til at isolere levedygtige MDSC populationer er vigtige til at belyse de forskellige molekylære mekanismer for undertrykkelse in vitro og in vivo. Nylig Ghansah gruppe har rapporteret om en udvidelse af MDSC i en murin bugspytkirtelkræft model. Vores tumorbærende MDSC viser et tab af homeostase og øget suppressive funktion sammenlignet med naive MDSC 13. MDSC procentdele er signifikant mindre i lymfoide rum af naive versus tumor-bærende mus. Dette er en væsentlig hage, som ofte forhindrer nøjagtig komparative analyser af disse MDSC. Derfor berige Gr-1 + leukocytter fra naive mus forud for fluorescensaktiveret cellesortering (FACS) forøger renheden, levedygtighed og reducerer sortering tid. Imidlertid berigelse af Gr-1 + leukocytter fra tumorbærende mus er valgfri, da disse er i overflod til hurtig FACS-sortering. Derfor, i denne protokol, beskriver vi en meget effektiv metode til immunfænotypebestemmelse MDSC og berigende Gr-1 + leukocytter fra milt fra naive mus til sortering MDSC i tide. Immunokompetente C57BL / 6 mus inokuleret med murin Panc02 ceLLS subkutant henviser naive mus modtager 1XPBS. Ca. 30 dage efter inokulering, milten høstes og behandles til enkelt-cellesuspensioner ved anvendelse af en celledissociering sigte. Splenocytter derefter røde blodlegemer (RBC) blev lyseret, og en aliquot af disse leukocytter farves med fluorochrom-konjugerede antistoffer mod Mac-1 og Gr-1 immunofænotype MDSC procentdele anvendelse af flowcytometri. I et parallelt eksperiment hele leukocytter fra naive mus farvet med fluorescenskonjugerede Gr-1-antistoffer og inkuberet med PE-mikrokugler og positivt selekteret ved hjælp af en automatiseret magnetisk aktiveret cellesortering (autoMACS) Pro Separator. Dernæst bliver en portion af Gr-1 + leukocytter farvet med Mac-1-antistoffer til at identificere forøgelse MDSC procentdele anvendelse af flowcytometri. Dag er disse GR1 + berigede leukocytter er klar til FACS-sortering af MDSC skal anvendes i komparative analyser (naive vs tumorbærende) i in vivo og in vitro </em&gt; assays.

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

Dette er en hurtig og omfattende fremgangsmåden immunfænotypebestemmelse Myeloide afledte suppressorceller (MDSC) og berige Gr-1<sup&gt; +</sup&gt; Leukocytter fra mus milte. Denne metode anvender flowcytometri og AutoMACS Cell sortering for at berige for levedygtige Gr-1<sup&gt; +</sup&gt; Leukocytter før FACS-sortering af MDSC til anvendelse<em&gt; In vivo</em&gt; Og<em&gt; In vitro</em&gt; Assays.

Fremstilling af myeloide afledte suppressorceller (MDSC) fra naive og pancreas Tumorbærende mus under anvendelse af flowcytometri, og Automatiseret magnetisk aktiveret cellesortering (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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