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 erkjenner USF flowcytometrisystemer Kjerne Facility. Vi ønsker å takke Dr. Denise Cooper for å dele ressurser. Vi ønsker også å takke Maya Cohen, Laura Pendleton og Diana Latour for deres hjelp i å sette opp og filmingen av denne videoen. NN støttet av NSF FG-LSAMP Bridge til doktorgrad Fellowship HRD # 0929435. Dette arbeidet ble finansiert av American Cancer Society Institutional Research Grant # 93-032-13/Moffitt Cancer Center tildelt TG.

Før du starter, forberede følgende løsninger: 3% Farging Media (SM): -3% Fetal bovin serum (FBS) i 1X fosfatbuffer saltvann (PBS) MACS Buffer (MB): - 0,5% Albumin fra Bovine Serum (BSA) i 1XPBS 1. Harvests spleens fra Mus <ol><li> Subkutant injisere 6-8 uker av alder C57BL / 6 mus (Harlan) med 1,5 x 10 5 murine Panc02 celler suspen...

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Dette er en detaljert metode for å behandle og immunophentyping MDSC populasjoner som gjelder for ulike lymfevev fra ulike dyremodeller. Spesielt kan autoMACS berikelse brukes til isolering av ulike leukocytter populasjoner inkludert Gr-1 uttømming av splenocytes 4, rensing av myeloide undergrupper fra splenocytes og lymfeknuter 5, isolering av benmarg nøytrofile 14 og rensing av CD8 + T celler fra milt og lymfeknuter 15. Uavhengig av cellen befolkning av interes...

<table border="1"><tbody><tr><td> REAGENT </td><td> COMPANY </td><td> Catalog # </td><td> KOMMENTARER </td></tr><tr><td> 1X Fosfatbufret Saline </td><td> Thermo Scientific Hyclone </td><td> SH30028.02 </td><td> Ca 2 + / Mg 2 + / Phenol Red-fri </td></tr><tr><td> Albumin fra Bovine Serum (BSA) </td><td> Sigma-Aldrich </td><td> A7906 </td><td> La BSA oppløse uforstyrret i PBS, sterilt miljø </td></tr><tr><td> Fetal bovin serum (FBS) </td><td> Thermo Scientific Hyclone </td><td> SV3001403HI </td><td> Heat Inaktivert; sterilt miljø </td></tr><tr><td> Rat anti-mus CD16/32 monoklonalt antistoff (Fc Block) </td><td> BD Biosciences </td><td> 553142 </td><td> Sterilt miljø </td></tr><tr><td> Anti-mus CD11b (Mac-en) 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> Sterilt miljø </td></tr><tr><td> Anti-mus Ly6G (Gr-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> Sterilt miljø </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> Serial Fortynning sterilt miljø </td></tr><tr><td> Cell Dissosiasjon Sieve </td><td> Sigma-Aldrich </td><td> CD1-1KT </td><td> Autoklav før bruk </td></tr><tr><td> 70-mikrometer sil </td><td> BD Biosciences </td><td> 352350 </td><td> Sterilt miljø </td></tr><tr><td> 1X RBC Buffer </td><td> eBiosciences </td><td> 00-4333-57 </td><td> Varm til romtemperatur før bruk, sterilt miljø </td></tr><tr><td> Petriskåler </td><td> Fisher Scientific </td><td> 08-757-12 </td><td> Sterilt miljø </td></tr><tr><td> 50ml conke rør </td><td> Thermo Scientific </td><td> 339652 </td><td> Sterilt miljø </td></tr><tr><td> 5 ml 12X75mm polystyren rund bunn rør </td><td> BD Biosciences </td><td> 352054 </td><td> Kjent som FACS rør; sterilt miljø </td></tr><tr><td> 96-brønn V-bunnplater </td><td> Corning </td><td> 3897 </td><td> Sterilt miljø </td></tr><tr><td> Trypan Blå </td><td> Cellgro </td><td> 25-900-CI </td><td> Sterilt miljø </td></tr><tr><td> PE MicroBeads </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> Sterilt 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 befolkning umodne makrofager, dendrittiske celler og granulocytter som akkumuleres i lymfoide organer i patologiske tilstander, inkludert parasittisk infeksjon, betennelse, traumatisk stress, graft-versus-vert sykdom, diabetes og kreft 1-7. Hos mus, MDSC ekspress Mac-en (CD11b) og Gr-1 (Ly6G og Ly6C) overflateantigener 7. Det er viktig å merke seg at MDSC er godt studert i ulike tumor-bærende vertene hvor de er betydelig utvidet og undertrykke anti-tumor immunresponser sammenlignet med naive kolleger 7-10. Men avhengig av patologisk tilstand, er det forskjellige subpopulasjoner av MDSC med forskjellige mekanismer og delmål for undertrykkelse 11,12. Derfor, effektive metoder for å isolere levedyktige MDSC bestander er viktig i å forklare deres ulike molekylære mekanismer for undertrykkelse in vitro og in vivo. Nylig, Ghansah gruppe har rapportert utvidelsen av MDSC i en murine bukspyttkjertelkreft modell. Vår tumor-bærende MDSC viser et tap av homeostase og økte undertrykkende funksjon i forhold til naive MDSC 13. MDSC prosenter er betydelig mindre i lymfoide avdelinger av naive vs tumor-bærende mus. Dette er en stor forbeholdet, som ofte hindrer nøyaktige komparative analyser av disse MDSC. Derfor berikende Gr-1 + leukocytter fra naive mus før Fluorescence Activated Cell Sorting (FACS) forbedrer renhet, levedyktighet og reduserer sortere tid. Imidlertid er anrikning av Gr-1 + leukocytter fra tumor-bærende mus valgfri da disse er i overflod for raske FACS sortering. Derfor, i denne protokollen, beskriver vi en svært effektiv metode for immunfenotyping MDSC og berikende Gr-1 + leukocytter fra spleens av naive mus for sortering MDSC på en riktig måte. Immunkompetente C57BL / 6 mus er inokulert med murine Panc02 ceLLS subkutant mens naive mus motta 1XPBS. Ca 30 dager etter inokulasjon; spleens høstes og bearbeides til encellede suspensjoner ved hjelp av en celle dissosiasjon sil. Splenocytes blir så røde blodceller (RBC) lysert og en delmengde av disse leukocytter er farget med fluorokromkonjugerte konjugerte antistoffer mot Mac-en og Gr-1 til immunophenotype MDSC prosenter ved hjelp av flowcytometri. I en parallell eksperiment, blir hele leukocytter fra naive mus farget med fluorescerende-konjugerte Gr-1 antistoffer, ruges med PE-MicroBeads og positivt valgt ved hjelp av en automatisert Magnetic Aktivert Cell Sorting (autoMACS) Pro Separator. Deretter blir en delmengde av GR-1 + leukocytter farget med Mac-1 antistoffer for å identifisere økningen i MDSC prosenter ved hjelp av flowcytometri. Nå, disse GR1 + beriket leukocytter er klare for FACS sortering av MDSC skal brukes i komparative analyser (naivt vs tumor-bærende) i in vivo og in vitro </em&gt; analyser.

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 rask og omfattende metode for immunfenotyping myelogen Avledede suppressor celler (MDSC) og berikende Gr-1<sup&gt; +</sup&gt; Leukocytter fra mus spleens. Denne metoden bruker flowcytometri og AutoMACS Cell Sortering å berike for levedyktige Gr-1<sup&gt; +</sup&gt; Leukocytter før FACS sortering av MDSC for bruk<em&gt; In vivo</em&gt; Og<em&gt; In vitro</em&gt; Analyser.

Utarbeidelse av myeloide Bearbeidede suppressor celler (MDSC) fra naiv og bukspyttkjertelen Tumor-bærende mus med flowcytometrisystemer og Automated Magnetic Aktivert Cell Sorting (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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