Name: an in vitro model to study heterogeneity of human macrophage differentiation and polarization
Uploaded: 2013-06-12T13:00:00
Description: Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

Vi takker Nadine Wambsganss for fremragende teknisk bistand. Dette arbejde blev støttet af en bevilling fra Deutsche Forschungsgemeinschaft (GL599/1-1) og dels af en bevilling fra Fornyelsesfonden FRONTIER (University of Heidelberg) til CAG

1.. Protokol <ol><li> Forbered Buffere som følger: <ol><li> Forbered buffer for PBMC isolation: &quot;Vask buffer&quot; = 0,02% EDTA i PBS (brug 0,5 M EDTA). </li><li> Forbered buffer for monocyt isolation: &quot;MACS skyllepuffer&quot; = 0,5% BSA (250 mg) + 2 mM EDTA (200 ul) + PBS (50 ml). Afgasse bufferen. </li><li> Forbered buffer for FACS-farvning og opbevaring af celler: &quot;FACS buffer&quot; = 10% FCS i PBS og &quot;fiksering buffer&quot; = 1% PFA i PBS. </li></ol></li><li> Trække 30 ml fuldblod fra en unde...

<ol>
	<li>Gordon, S., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+and+macrophage+heterogeneity.">Monocyte and macrophage heterogeneity.</a> Nat. Rev. Immunol. 5, 953-964 (2005).</li><li>Gordon, S., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+plasticity+of+mononuclear+phagocytes.">Diversity and plasticity of mononuclear phagocytes.</a> European Journal of Immunology. 41, 2470-2472 (2011).</li><li>Martinez, F. O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front. Biosci. 13, 453-461 (2008).</li><li>Ingersoll, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gene+expression+profiles+between+human+and+mouse+monocyte+subsets.">Comparison of gene expression profiles between human and mouse monocyte subsets.</a> Blood. 115, 10-19 (2010).</li><li>Gleissner, C. A., Sanders, J. M., Nadler, J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+aldose+reductase+during+foam+cell+formation+as+possible+link+among+diabetes,+hyperlipidemia,+and+atherosclerosis.">Upregulation of aldose reductase during foam cell formation as possible link among diabetes, hyperlipidemia, and atherosclerosis.</a> Arterioscler. Thromb. Vasc. Biol. 28, 1137-1143 (2008).</li><li>Gleissner, C. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+il-23-producing+type+1+macrophages+promote+but+il-10-producing+type+2+macrophages+subvert+immunity+to+(myco)bacteria.">Human il-23-producing type 1 macrophages promote but il-10-producing type 2 macrophages subvert immunity to (myco)bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 4560-4565 (2004).</li><li>Rey-Giraud, F., Hafner, M., Ries, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+monocyte-derived+macrophages+under+serum-free+conditions+improves+their+tumor+promoting+functions.">In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.</a> PLoS One. 7, e42656 (2012).</li><li>Boyle, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+intraplaque+hemorrhage+evokes+a+novel+atheroprotective+macrophage+phenotype.">Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.</a> American Journal of Pathology. 174, 1097-1108 (2009).</li><li>Cho, H. 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Monocytafledte makrofager repræsenterer den centrale celletype af det medfødte immunsystem. De spiller en vigtig rolle i mange inflammatoriske sygdomme, herunder åreforkalkning eller kræft 2.. Således studere makrofag biologi er vigtig for at øge vores viden om patofysiologien af ​​mange sygdomme og muliggøre udviklingen af ​​nye behandlingsformer. Mange undersøgelser gælder for musemodeller overudtrykker eller mangler visse gener af interesse. I tilfælde af mono...

Monocytafledte makrofager udgør en vigtig cellulær komponent af det medfødte immunsystem og bidrager til mange akutte eller kroniske inflammatoriske processer 1.. Makrofager spiller en vigtig rolle i mange inflammatoriske sygdomme som åreforkalkning eller kræft 2. Makrofager udviser en høj grad af plasticitet og er i stand til at antage forskellige fænotyper afhængigt af den lokale micromilieu 3.. Således studere makrofag differentiering og heterogenitet er vigtig for at øge vores viden om patofysiologien af ​​mange sygdomme og for at tillade identifikation af nye terapeutiske mål og udvikling af nye behandlingsformer. I mange tilfælde er murine modeller anvendt til at undersøge patofysiologien af ​​specifikke sygdomme. Men studerer makrofag biologi hjælp musemodeller ledsaget af flere mangler: (1) Den andel af leukocyt delmængdebetingelser numre (dvs. monocytter og granulocytter) i peripHeral blod af mus eller mennesker adskiller sig væsentligt tyder forskellige roller monocytter i murine og humane patofysiologi. (2) Der er betydelige forskelle i genekspression mellem murine og humane perifere blod monocytter tyder væsentlige forskelle i deres funktion under sundhed og sygdom 4.. (3) En række markører, der bruges til at identificere murine monocytter og makrofager (F4/80, Lyc osv.) Findes ikke i humane myeloide celler, hvilket gør overførslen af resultater i musemodeller for den menneskelige situation, temmelig vanskelig. Således, for at øge vores forståelse af makrofag differentiering og forskelligartethed i human sygdom, er vi nødt til at gøre brug af modeller arbejder med humane makrofager. Derfor, vi her beskrive en model af humant primær makrofag generation, er nem at bruge og tillader undersøgelse af humane monocyt-afledte makrofager in vitro under forskellige betingelser resulterer i forskellige makrofag popolarisation typer. I flere studier har vi brugt in vitro model for monocytafledte primære humane makrofager til at analysere makrofag biologi og dens potentielle relevans for human åreforkalkning 5-7. Selvom ikke fuldt ud erstatte forsøg med dyr eller humane væv opnået post mortem, modellen er beskrevet her tillader identifikation og validering af sygdomsmekanismer og terapeutiske mål, der kan være yderst relevant for forskellige menneskelige sygdomme.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube (sterile)</td>
			<td>Fisher</td>
			<td>055398</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS (1X), liquid (no calcium or magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>T9285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8806</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Human Monocyte Enrichment Kit</td>
			<td>StemCell Technologies</td>
			<td>19059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Macrophage-SFM (1X)</td>
			<td>Invitrogen</td>
			<td>12065-074</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P-4458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human M-CSF 10 &mu;g</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Culture Plates 6-well</td>
			<td>Fisher</td>
			<td>07-200-80</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

Monocytafledte makrofager udgør en vigtig celletype af det medfødte immunsystem. Musemodeller studerer makrofag biologi lider fænotypiske og funktionelle forskelle mellem murine og humane monocyt-afledte makrofager. Derfor har vi her beskrive en in vitro-model til at generere og studere primære humane makrofager. Kort fortalt, efter densitetsgradientcentrifugering af perifert blod udtaget fra en underarm vene, er monocytter isoleret fra perifere mononukleære celler ved anvendelse af negativ magnetisk perle isolation. Disse monocytter dyrkes derefter i seks dage under særlige betingelser at inducere forskellige typer af makrofag differentiering eller polarisering. Modellen er let at bruge og omgår problemerne med artsspecifikke forskelle mellem mus og menneske. Desuden er det tættere på in vivo end anvendelsen af immortaliserede cellelinier. Konklusionen er, at modellen er beskrevet her er egnet til at studere macrophage biologi, identificere sygdomsmekanismer og nye terapeutiske mål. Selvom ikke fuldt ud erstatte forsøg med dyr eller humane væv opnået post mortem, modellen er beskrevet her tillader identifikation og validering af sygdomsmekanismer og terapeutiske mål, der kan være yderst relevant for forskellige menneskelige sygdomme.

Anvendelse af protokollen beskrevet ovenfor, vi rutinemæssigt opnå 25,1 x 10 6 ± 2,2 x 10 6 monocytes/100 ml blod (gennemsnit ± standardafvigelse fra 26 uafhængige forsøg, figur 1A). Monocyt renhed som bestemt ved flowcytometrisk farvning for CD14 er rutinemæssigt større end 95% (97,1 ± 0,4%, gennemsnit ± standardafvigelse fra 3 uafhængige eksperimenter, figur 1B). Cellelevedygtighed af frisk isolerede monocytter som bestemt ved trypanblåt-farvning er r...

Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

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

Monocytafledte makrofager er vigtige celler i det medfødte immunsystem. Her beskriver vi en nem at bruge<em&gt; In vitro</em&gt; Model til at generere disse celler. Brug gradientcentrifugering, negativ perle isolation og særlige celledyrkningsbetingelser, kan monocytafledte makrofager Der genereres fænotypiske og funktionelle studier.

En<em&gt; In vitro</em&gt; Model til Studieinformationen Heterogeneity for Human Makrofag Differentiering 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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In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overekspression og oprensning af mennesket<em&gt; Cis</em&gt; -prenyltransferase i<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Opdigte optisk kvalitet glasoverflader for at studere makrofag Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

In Vitro Differentiering af mus granulocyt-makrofag-koloni-stimulerende faktor (GM-CSF)-producerer T hjælper (THGM) celler

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...