Name: isolation of mouse lung dendritic cells
Uploaded: 2011-11-22T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Forfatterne vil gerne takke for at Marilyn Dietrich på LSU Flowcytometri Core facilitet for hendes hjælp med cellesortering og Peter Mottram for hans hjælp med microphotographs. Dette arbejde blev finansieret af Flight Attendant Medical Research Institute, LSU-kompetitive forskning Program Award, og NIH / NIAID Tilskud P20 RR020159 og R03AI081171.

1. Lunge perfusion og enkelt celle suspension <ol><li> Aflive musen med en intraperitoneal injektion af ketamin / xylazin bedøvelse blanding (i mg / mus ketamin 1,8, xylazin 0,19) og afblødning via collum vene </li><li> Expose brysthulen ved at klippe og forsigtigt at trække tilbage den ydre hud af bughinden. Fortsæt at åbne mellemgulvet ved at skære i brystkassen til at afsløre både hjerte og lunger. </li><li> Perfuse forsigtigt lungerne ved hjælp af en 5 ml sprøjte fyldt med EDTA-HBSS (1 mm EDTA i calcium ...

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Isolering af pulmonal musen DC er en vigtig teknik til studiet af en bred vifte af respiratoriske stimuli. Processen med at opnå disse celler indeholder vigtige skridt, der forhindrer tab af celler samt cellernes levedygtighed og renhed. Perfusing lungen før kollektionen vil medvirke til at fjerne enhver perifere celler samt reducere forurenende erytrocytter. Brugen af ​​automatiserede dissociation kan advanteogus, når et stort antal lunger håndteres, ellers kan du vælge den alternative protokol holde sig for ?...

<table width="200" border="1"><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer (valgfri) </td></tr><tr><td> ACK Lysing buffer </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-mus CD11c (HL3) </td><td> BD Pharmingen </td><td> 5580979 </td><td> PE-Cy7 konjugeret </td></tr><tr><td> Anti-mus IA / IE (269) </td><td> BD-Pharmingen </td><td> 553623 </td><td> FITC konjugeret </td></tr><tr><td> Collangenase Type 1A </td><td> Sigma </td><td> 9891-500mg </td><td></td></tr><tr><td> Cell filtre </td><td> BD Falcon </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) mikrokugler </td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNase Jeg </td><td> Sigma </td><td> D5025-150KU </td><td></td></tr><tr><td> Hank er Balanced Salt-løsning </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> HEPES bufferopløsning </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Petriskåle 60 mm </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C rør </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> Gentle MACS dissociator </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS-Pro ™ </td><td> Miltenyi </td><td> 130-092-545 </td><td></td></tr><tr><td> FASCS Aria </td><td> BD </td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

Lung dendritiske celler (DC) spiller en fundamental rolle i sensing invaderende patogener 1,2 samt i kontrollen af tolerogenic svar 3 i luftvejene. Mindst tre delmængder af lunge dendritiske celler er blevet beskrevet i mus: konventionelle DC (CDC) 4, plasmacytoid DC (PDC) 5 og IFN-producerende killer DC (IKDC) 6,7. CDC delmængde er den mest fremtrædende DC delmængde i lungerne 8. Den fælles markør kendt for at identificere DC delmængder er CD11c, en type I transmembrane integrin (β2), som også er udtrykt på monocytter, makrofager, neutrofile og nogle B-celler 9. I nogle væv, bruger CD11c som markør for at identificere musen DC gyldig, som i milten, hvor de fleste CD11c + celler repræsentere CDC delmængde, der udtrykker høje niveauer af de store histocompatibility komplekse klasse II (MHC-II). Men lungen er en mere heterogen væv, hvor derside DC delmængder, der er en høj procentdel af en særskilt cellepopulation, der udtrykker høje niveauer af CD11c anfald lave niveauer af MHC-II. Baseret på at karakterisere og for det meste på sin udtryk for F4/80, en milt makrofag markør, har CD11c hi MHC-II lo lunge cellepopulation blevet identificeret som pulmonale makrofager 10 og senere, som en potentiel DC forløber 11. I modsætning til mus PDC, har studiet af den særlige rolle, CDC i det pulmonale immunforsvar været begrænset på grund af manglen på en specifik markør, der kunne hjælpe i isolation af disse celler. Derfor, i dette arbejde, beskriver vi en procedure for at isolere højtrenset musen lunge CDC. Isolering af pulmonale DC delmængder repræsenterer et meget nyttigt redskab til at opnå indsigt i funktionen af ​​disse celler som reaktion på respiratoriske patogener såvel som miljømæssige faktorer, der kan udløse værtens immunrespons i lungerne.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

En højtrenset forberedelse af musen lunge dendritiske celler er beskrevet. Særlig vægt gives til isolering af konventionelle dendritiske celler delmængde.

Isolering af Mouse Lung dendritiske celler

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Research

JoVE Journal

Immunology and Infection

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Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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Præcision-cut muselunge Skiver at visualisere Levende Pulmonal dendritiske celler

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