Name: depletion and reconstitution of macrophages in mice
Uploaded: 2012-08-01T13:00:00
Description: Watch this Scientific Journal Video about Depletion and Reconstitution of Macrophages in Mice at JoVE.com

Dette arbejde blev understøttet af en &quot;Grant i støtte for forskning&quot; fra Crohns og Colitis Foundation of Canada til LMS, der understøttes af en canadisk Association of Gastroenterology / Canadian Association of Health Research / Crohns og Colitis Foundation of Canada New Investigator Award.

1. Ozonlagsnedbrydende Makrofager Brug Clodronat-holdige liposomer <ol><li> Liposomer opbevares ved 4 ° C. To timer forud for injektion, fjernes clodronat-holdige liposomer, PBS-holdige liposomer, eller sterilt PBS (injektion kontrol) fra køleskabet for at tillade dem at akklimatisere sig til stuetemperatur (18 ° C). </li><li> Vend rørene indeholdende liposomer 8-10 gange for at sikre en jævn fordeling før lastning 200 pi i en 1 ml sprøjte. Vedhæfte en 26 gauge nål til toppen af ​​sprøjten. </li><li> Bru...

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	<li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3, 23-35 (2003).</li><li>Rescigno, M., Lopatin, U., Chieppa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+among+dendritic+cells,+macrophages,+and+epithelial+cells+in+the+gut:+implications+for+immune+tolerance.">Interactions among dendritic cells, macrophages, and epithelial cells in the gut: implications for immune tolerance.</a> Curr. Opin. Immunol. 20, 669-675 (2008).</li><li>Heinsbroek, S. 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Immunol. 178, 4557-4566 (2007).</li><li>van Rooijen, N., Hendrikx, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+for+specific+depletion+of+macrophages+from+organs+and+tissues.">Liposomes for specific depletion of macrophages from organs and tissues.</a> Methods Mol. Biol. 605, 189-203 (2010).</li><li>Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+vitreal+macrophages+and+circulating+leukocytes+in+retinal+neovascularization.">The roles of vitreal macrophages and circulating leukocytes in retinal neovascularization.</a> Invest. Ophthalmol. Vis. Sci. 52, 1431-1438 (2011).</li><li>Weisser, S. 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Makrofager er fagocytiske celler, som spiller en vigtig rolle i immunsystemet. De er ansvarlige for at indlede den medfødte immunreaktion og lede den erhvervede immunreaktion. Typisk er klassisk aktiverede makrofager aktiveret ved IFNy eller LPS og er ansvarlige for fjernelse patogener og montering et inflammatorisk respons 1. Omvendt er alternativt aktiverede makrofager aktiveret med IL-4 eller IL-13 og spiller en rolle i debris skylning og vævsremodellering under opløsning af inflammation 1. S...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Clodronat-holdige liposomer </td><td> <a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a> </td><td></td></tr><tr><td> PBS-indeholdende liposomer </td><td> <a href="http://Clodronateliposomes.org" target="_blank">Clodronateliposomes.org</a> </td><td></td></tr><tr><td> Sterilt PBS pH 7,4 </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> IMDM </td><td> Invitrogen </td><td> 12440 </td></tr><tr><td> Føtalt kalveserum (FCS) </td><td> Invitrogen </td><td> 12483 </td></tr><tr><td> eddikesyre </td><td> BDH ARISTAR </td><td> BDH3092-500MLP </td></tr><tr><td> Monothioglycerol (MTG) </td><td> Sigma </td><td> 96-275 </td></tr><tr><td> Recombinant murine MCSF </td><td> StemCell Technologies </td><td> 02951 </td></tr><tr><td> Rekombinant murin GM-CSF </td><td> StemCell Technologies </td><td> 02935 </td></tr><tr><td> Rekombinant murin IL-3 </td><td> StemCell Technologies </td><td> 02903 </td></tr><tr><td> Rekombinant murin IFN-y </td><td> StemCell Technologies </td><td> 02746 </td></tr><tr><td> Rekombinant murin IL-4 </td><td> StemCell Technologies </td><td> 02714 </td></tr><tr><td> Celledissociering Buffer </td><td> Invitrogen </td><td> 13150-016 </td></tr><tr><td> Rat anti-F4/80 Antistof </td><td> ABD Serotec </td><td> MCA497GA </td></tr><tr><td> Muse anti-Argi antistof </td><td> BD Transduction Laboratories </td><td> 610708 </td></tr></tbody></table> Tabel 1. Specifikke reagenser, der anvendes i denne protokol.

depletion and reconstitution of macrophages in mice

Makrofager er kritiske aktører i den medfødte immunrespons til infektiøs udfordring eller skade, indlede det medfødte immunforsvar og lede den erhvervede immunreaktion. Makrofag dysfunktion kan føre til en manglende evne til at montere en passende immunrespons, og som sådan er blevet impliceret i mange sygdomsforløb, herunder inflammatoriske tarmsygdomme. Makrofager vise polariserede fænotyper, der er groft inddeles i to kategorier. Klassisk aktiverede makrofager, aktiverede ved stimulering med IFNy eller LPS, spiller en vigtig rolle som reaktion på bakteriel provokation henviser alternativt aktiverede makrofager, aktiveret med IL-4 eller IL-13 deltager i debris skylning og vævsremodellering og har været impliceret i opløsning fase inflammation. Under en inflammatorisk reaktion in vivo, er makrofager ligger midt en kompleks blanding af infiltrerende immunceller og kan deltage i forværre eller resolving inflammation. For at definere den rolle, makrofager in situ i et helt dyr model, er det nødvendigt at undersøge effekten af ozonlagsnedbrydende makrofager fra komplekst miljø. At stille spørgsmål om den rolle, makrofag fænotype in situ, kan fænotypisk definerede polariserede makrofager udledes ex vivo, fra knoglemarvsaspirater og sat tilbage til mus, med eller uden forudgående udtynding af makrofager. I protokollen præsenteret her clodronat-holdige liposomer, versus PBS injicerede kontroller blev anvendt til at udtømme colon makrofager under dextrannatriumsulfat (DSS)-induceret colitis hos mus. Desuden blev polariserede makrofager afledt ex vivo og overført til mus ved intravenøs injektion. En advarsel til denne fremgangsmåde er, at clodronat-holdige liposomer udtømme alle professionelle fagocytter, herunder både dendritiske celler og makrofager, så for at sikre den effekt observeret ved nedbrydningen er makrofag-specifik, rekonstitution of fænotype ved adoptiv overførsel af makrofager er nødvendig. Systemisk makrofag udtømning hos mus kan også opnås ved tilbagekrydsning mus på en CD11b-DTR baggrund, som er en fremragende komplementær tilgang. Den fordel, clodronat-holdig liposomdispersions-medieret nedbrydning er, at det ikke kræver tid og de omkostninger der er involveret i tilbagekrydsning mus, og det kan anvendes i mus, uanset om baggrunden for mus (C57BL / 6, BALB / c eller blandet baggrund ).

Watch this Scientific Journal Video about Depletion and Reconstitution of Macrophages in Mice at JoVE.com

Depletion and Reconstitution of Macrophages in Mice

Makrofager spiller en central rolle i homeostase og patologi i mange væv. Protokollen præsenteres her beskriver metoder til nedbrydende makrofager<em&gt; In vivo</em&gt; Afledt polariserede makrofager fra knoglemarvsaspirater, og adoptivt overfører makrofager i mus. Disse teknikker tillader bestemmelse af den rolle, polariserede makrofager spiller i sundhed og sygdom.

Udtømning og Rekonstituering af makrofager i mus

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Research

JoVE Journal

Immunology and Infection

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Study of Phagolysosome Biogenesis in Live Macrophages

Undersøgelse af fagolysosomet Biogenese i Live makrofager

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Anvendelse af<em&gt; Shigella flexneri</em&gt; For at studere autofagi-Cytoskeleton interaktioner

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

Proteomisk Profilering af makrofager ved 2D elektroforese

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Makrofag Kolesterol Udtømmelse og dens indflydelse på fagocytose af<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Kvantificering af Cytosolisk vs. vakuolær<em&gt; Salmonella</em&gt; I Primære Makrofager af Differential Permeabilisering

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolering af Salmonella typhimurium-indeholdende Phagosomes fra makrofager

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃƒÅ½Ã‚Â³R Cross-linking

Cytometric flowmåling af ROS produktion i makrofager i svar til FcγR tværbinding

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Kvantificering af Monoyte Chemotaktisk aktivitet in vivo og karakterisering af blod monocytter afledt makrofager

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...