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 arbeidet ble støttet av American Heart Association (BGIA 7.850.037 til Dr. Beiyan Zhou).

En. Isolering av benmargceller <ol><li> Isoler femur og tibia bein 6-8 uker gamle mus, skyll av hår og deretter kuttet åpne beinet. </li><li> Bruk en 21G nål og 10 ml sprøyte til å skylle ut margen i kaldt PBS 2% varmeinaktivert Fetal Bovine Serum (FBS) (3-5 ml / mus). </li><li> Pass marg gjennom en 21G nål 4-6 ganger for å distansere cellene. </li><li> Passere cellene gjennom en 70 mikrometer celle sil for å fjerne celle klumper, bein, hår og andre celler / vev. </li><li> Tilsett 3 volum av NH4Cl-løsni...

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Vi rapporterer her en enkel og lett tilpasses in vitro prosedyre for å indusere aktivering av makrofager som stammer fra benmargsstamceller. Denne prosedyre kan benyttes for undersøkelse av mekanismene som er ansvarlige for polarisering av makrofager. Renheten av modne makrofager innhentet ved hjelp av denne protokollen gjennomsnitt 95-99%, og ingen ekstra rensing må utføres. For å undersøke funksjonen av spesifikke gener av interesse i sammenheng med makrofag polarisering, ektopisk ekspresjon eller genet...

Forskjellig fra klassiske inflammatoriske responser, makrofager som infiltrerer vev ofte vise polarisert aktivering status som spiller en avgjørende rolle i å regulere vert vev fysiologiske funksjoner 1-8. Ved stimulering, makrofag aktivering kan bli sortert i klassiske (M1) og alternativt (M2) aktivering 2, 4, 9. M1 makrofag aktivering avhenger av Toll-lignende reseptorer (TLR) og aktivering av kjernefysiske faktor kappa B (NFκB) / c-Jun N-terminal kinase 1 (JNK1), som fører til produksjon av inflammatoriske cytokiner, for eksempel TNF-α og IL- 1β og aktivering av iNOS som resulterer i økt produksjon av reaktive oksygenforbindelser slik som nitride oksyd (NO), 10, 11.. I kontrast, M2 makrofag aktivering rekrutter PPARγ, PPARδ, eller IL-4-STAT6 pathways, som fører til alternative, anti-inflammatoriske (M2) aktivering som er forbundet med oppregulering av mannose-reseptoren CD206, og arginase 1 (Arg1) 6, 12 - 14 &lt;/ Sup&gt;. Benmarg avledet makrofager (BMDM) presenterer en ideell in vitro modell for å forstå mekanismene som styrer polarisering av aktiverte makrofager 15. Nærmere bestemt, kan aktivering av makrofager M1 bli indusert av lipopolysaccharides (LPS) stimulering, mens polarisering av M2 makrofager kan induseres ved IL-4 og / eller IL-13. Modne benmarg avledet makrofager og aktiverte makrofager kan identifiseres gjennom flowcytometri analyse for uttrykk for overflateantigenene, inkludert CD11b, F4/80, CD11c, CD206, CD69, CD80 og 9 CD86, 16, 17. I tillegg kan forandringer i celle-cytokinproduksjon og signalveier assosiert med makrofag polarisering bli målt ved kvantitativ RT-PCR og western blotting, henholdsvis. Oppsummert kan mus benmarg avledet makrofager fungere som en relevant modell for å studere macrophage 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

Artikkelen beskriver et lett enkelt adaptiv in vitro modell for å undersøke makrofag polarisering. I nærvær av GM-CSF/M-CSF, blir hematopoetiske stamceller / stamceller fra benmargen rettet inn i monocyttisk differensiering, etterfulgt av M1 eller M2 stimulering. Aktiveringsstatusen kan spores av endringer i celleoverflateantigener, genekspresjon og celle signalveier.

En skjematisk beskrivelse av BMDM generasjon prosedyren er presentert (figur 1). Høy renhet av modne makrofager kan observeres på dag 7 når de representerer 95-99% av CD11b + F4/80 +-celler (Figur 2). Polariserte makrofager kan undersøkes ved hjelp av antistoffer mot CD11b, F4/80, etterfulgt CD11c og CD206 ved flowcytometri analyse. Som vist i figur 3, er M1 makrofager oppdaget som CD11b + F4/80 + CD11c + CD206-celler (Q2), mens M2 makrofager er CD11b + F4/80 + CD11...

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

Artikkelen beskriver et lett enkelt adaptiv<em&gt; In vitro</em&gt; Modell for å undersøke macrophage polarisering. I nærvær av GM-CSF/M-CSF, blir hematopoetiske stamceller / stamceller fra benmargen rettet inn i monocyttisk differensiering, etterfulgt av M1 eller M2 stimulering. Aktiveringsstatusen kan spores av endringer i celleoverflateantigener, genekspresjon og celle signalveier.

Undersøkelse av Macrophage Polarisering Bruk Bone Marrow makrofager som stammer

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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Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

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

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

En<em&gt; In vitro</em&gt; Modell for å studere heterogenitet of Human Macrophage Differensiering og Polarisering

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

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Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

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Murine Hind Limb Long Bone Dissection og benmarg Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Generasjon og identifisering av GM-CSF Avledet Alveolære lignende makrofager og dendrittiske celler fra mus Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

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A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...