Name: investigation of macrophage polarization using bone marrow derived macrophages
Uploaded: 2013-06-23T14:00:00
Description: Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Dette arbejde blev støttet af American Heart Association (BGIA 7.850.037 til Dr. Beiyan Zhou).

1.. Isolering af knoglemarvsceller <ol><li> Isoler lårben og skinneben knogler 6-8 uger gamle mus, skylle håret og derefter skåret op knoglen. </li><li> Brug en 21G kanyle og 10 ml sprøjte til at skylle ud marv i koldt PBS +2% varmeinaktiveret føtalt bovint serum (FBS) (3-5 ml / mus). </li><li> Passere knoglemarv gennem en 21G nål 4-6 gange for at dissociere cellerne. </li><li> Pass celler gennem en 70 um cellefilter at fjerne celleklumper, knogler, hår og andre celler / væv. </li><li> Tilsæt 3 rumfang N...

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Vi rapporterer her en enkel og let tilpasses in vitro procedure til at inducere aktivering af makrofager afledt af knoglemarven. Denne procedure kan anvendes til undersøgelse af mekanismerne bag polarisering af makrofager. Renheden af ​​modne makrofager opnået ved hjælp af denne protokol gennemsnit 95-99%, og ingen yderligere rensning procedurer er påkrævet. At undersøge funktionen af ​​specifikke gener af interesse i forbindelse med makrofag polarisering, ektopisk udtryk eller genspecifik knockdo...

Adskilt fra klassiske inflammatoriske reaktioner, makrofager, der infiltrerer væv udviser ofte polariseret aktivering status, spiller en afgørende rolle i reguleringen af værtsvæv fysiologiske funktioner 1-8. Ved stimulering, kan makrofagaktivering sorteres i klassisk (M1) og alternativ (M2) aktivering 2, 4, 9. M1 makrofagaktivering afhænger Toll-lignende receptorer (TLRs) og aktivering af nuklear faktor kappa B (NFKB) / c-Jun N-terminal kinase 1 (Jnk1), hvilket fører til produktion af inflammatoriske cytokiner, såsom TNF-α og IL- 1β og aktivering af iNOS, som resulterer i øget produktion af reaktive ilt arter såsom nitrid-oxid (NO) 10, 11. I modsætning hertil fører M2 makrofagaktivering rekrutter PPARy, PPARδ eller IL-4-Stat6 veje, til alternativ, anti-inflammatorisk (M2) aktivering, som er forbundet med opregulering af mannosereceptoren CD206, og arginase 1 (arg1) 6, 12 - 14 &lt;/ Sup&gt;. Knoglemarv afledte makrofager (BMDM) udgør en ideel in vitro-model til at forstå de mekanismer, der styrer polarisering af aktiverede makrofager 15. Specifikt kan aktivering af M1 makrofager induceres af lipopolysaccharider (LPS) stimulering, mens polarisering af M2 makrofager kan induceres af IL-4 og / eller IL-13. Modne knoglemarv afledt makrofager og aktiverede makrofager kan identificeres gennem flowcytometrianalyse for ekspression af overflade-antigener, herunder CD11, F4/80, CD11c, CD206, CD69, CD80 og CD86 9, 16, 17. Desuden kan ændringer i cytokinproduktion og celle signalveje forbundet med makrofag polarisering måles ved kvantitativ RT-PCR og western blotting, hhv. Sammenfattende kan mus knoglemarv afledte makrofager tjene som en relevant model til at studere makrofag polarisering in vitro.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Thermo Scientific</td>
 <td>SH30259.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Invitrogen</td>
 <td>10438-026</td>
 <td></td>
 </tr>
 <tr>
 <td>Murine GM-CSF</td>
 <td>PeproTech</td>
 <td> 315-03</td>
 <td></td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>StemCell Technologies</td>
 <td>7850</td>
 <td></td>
 </tr>
 <tr>
 <td>L-929</td>
 <td>ATCC</td>
 <td>CCL-1</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>10 x PBS</td>
 <td>Thermo Scientific</td>
 <td>AP-9009-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b-APC</td>
 <td>eBioscience</td>
 <td>17-0112-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse F4/80-FITC</td>
 <td>eBioscience</td>
 <td>11-4801-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD69-PE</td>
 <td>eBioscience</td>
 <td>12-0691-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD86-PE</td>
 <td>eBioscience</td>
 <td>12-0862-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Propidium Iodine</td>
 <td>Invitrogen</td>
 <td>P3566</td>
 <td></td>
 </tr>
 </tbody>
</table>

investigation of macrophage polarization using bone marrow derived macrophages

I artiklen beskrives en let let adaptiv in vitro-model til at undersøge makrofag polarisering. I nærvær af GM-CSF/M-CSF er hæmatopoietiske stamceller / stamceller fra knoglemarven ledes ind monocytær differentiering, efterfulgt af M1 eller M2 stimulation. Aktiveringen status kan spores af ændringer i celleoverfladeantigener, genekspression og celle signalveje.

En skematisk beskrivelse af BMDM genereringen præsenteres (figur 1). Høj renhed af modne makrofager kan ses på dag 7, når de repræsenterer 95 til 99% af CD11 + F4/80 +-celler (figur 2). Polariserede makrofager kan undersøges under anvendelse af antistoffer mod CD11, F4/80, efterfulgt CD11 c og CD206 ved flowcytometri-analyse. Som vist i figur 3, er M1 makrofager detekteret som CD11 + F4/80 + CD11c + CD206-celler (Q2), hvorimod M2 makrofager er CD11 + F4/80 + CD11c...

Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

I artiklen beskrives en let let adaptiv<em&gt; In vitro</em&gt; Model til at undersøge makrofag polarisering. I nærvær af GM-CSF/M-CSF er hæmatopoietiske stamceller / stamceller fra knoglemarven ledes ind monocytær differentiering, efterfulgt af M1 eller M2 stimulation. Aktiveringen status kan spores af ændringer i celleoverfladeantigener, genekspression og celle signalveje.

Undersøgelse af Makrofag Polarization Brug knoglemarvsafledte makrofager

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

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