Diese Arbeit wurde unterstützt durch einen Zuschuss aus den Natur- und Ingenieurwissenschaften Research Council of Canada unterstützt (NSERC, gewähren keine 298.515-2011). AL ist der Empfänger eines Ph.D. Stipendium von den Natur- und Ingenieurwissenschaften Research Council of Canada (NSERC) und MS wurde aus einem Zuschuss von der kanadischen Institutes of Health Research (CIHR, gewähren keine. MOP123246).

Alle Verfahren wurden in Übereinstimmung mit dem Canadian Council on Animal Care Leitlinien nach Genehmigung durch die Institutional Animal Care Committee des Centre de recherche du Centre Hospitalier de l&#39;Université de Montréal (CRCHUM) durchgeführt. 1. Isolierung, Identifizierung und Kultur von Murine Peritonealmakrophagen <ol><li> Bereiten Sie 3,8% Brau Thioglykolat-Medium. Um dies zu tun, aussetzen 38 g Thioglykolat-Medium in 1000 ml destilliertem Wasser. Bringen Lösung zum Kochen bringen, um das Medium vollständig zu lösen. Sterilisieren durch Autoklavieren bei 121 ° C für 15 min. Speichern Sie bis zu 3 Monate in der Dunkelheit, bei RT 18. HINWEIS: Verwerfen, wenn Trübung entwickelt, die eine bakterielle Kontamination zeigt. Die Lösung kann bis zu einem Jahr im Dunkeln gehalten werden, wenn steril gehalten. </li><li> Verwendung von 1 ml-Spritzen bis 23 G Nadeln befestigt injizieren 1 ml 3,8% Brewer Thioglykolat-Medium in die Peritonealhöhle jeder Vereinbarungse und warten Sie für 3 Tage. Verwenden Sie neue Spritze und Nadel für jede Maus. </li><li> Betäuben Mäusen durch intraperitoneale Injektion von Natriumpentobarbital (100 mg / kg) und euthanize Mäuse durch Genickbruch. Überprüfen Sie die ordnungsgemäße Betäubung durch Überprüfung der Atemfrequenz. In der Regel schnelle Atmung zeigen, dass die Maus nicht tief anästhesiert. </li><li> Den Unterleib jeder Maus mit 70% Ethanol waschen. Mit einer Schere, führen Sie einen seitlichen Schnitt entlang der unteren Mittellinie des Bauchfells. </li><li> Mit einer Pinzette, ziehen Sie die Bauchhaut, um die transparente Bauchhaut freizulegen. Verwendung von 5 ml Spritzen an 20 G Nadeln befestigt injizieren 5 ml kaltem DPBS in die Peritonealhöhle jeder Maus. </li><li> Führen Sie eine sanfte Massage auf der Bauchhöhle und dann sorgfältig absaugen Flüssigkeit ohne Punktieren jedes Organ. Entfernen Sie die Nadel und verzichtet der Peritonealflüssigkeit in 50 ml konischen Zentrifugenröhrchen. </li><li> Zentrifugieren für 10 min bei 400 × g in einer gekühlten Zentrifuge.Zellen müssen während des gesamten Verfahrens kalt bleiben. Überstand verwerfen und resuspendieren Zellpellet in RPMI-Medium 1640. </li><li> Verwendung eines Hämozytometers zählen Zellen und einzustellen, um die Zelldichte auf 1 x 10 6 Zellen / ml. </li><li> Charakterisierung des Phänotyps isolierten Zellen durch Durchflusszytometrie unter Verwendung von 1 x 10 6 Zellen pro Maus und antibodie gegen F4 / 80 (eine Oberfläche Antigens, das auf Makrophagen). </li></ol> 2. Zellbehandlungen <ol><li> Direkt nach der Isolierung, fügen Sie 1 x 10 6 Zellen in jede Vertiefung. Lassen die murine peritoneale Makrophagen durch Kultivieren für 1 bis 2 Stunden bei 37 ° C in der 6-Well-Platten zu halten. Entfernen Sie nicht anhaftende Zellen durch vorsichtiges Waschen 3 mal mit warmem PBS. </li><li> Anschließend Kulturzellen in 900 &amp; mgr; l serumfreiem DMEM für 24 h in Gegenwart der folgenden TLR-Liganden: (Pam3CSK4-TLR1 / 2 (0,5 mg / ml), Poly (I: C) -TLR3 (10 mg / ml) ; LPS-TLR4 (100 ng / ml); Flagellin-TLR5 (100 ng / ml); FSL1- TLR6 / 2 (100 ng / ml); ssRNA40-TLR7 (1 ug / ml); ODN1826-TLR9 (1 uM). HINWEIS: Bereiten Sie für jeden Liganden ein 10-faches Konzentrat. 100 l des letzteren in jede Vertiefung, wodurch die Durchführung einer 10-fachen Verdünnung. </li></ol> 3. RNA-Isolierung <ol><li> Entfernen Sie alle mittleren und lysieren Zellen direkt in den sechs-Well-Platten durch Zugabe von 1 ml TRIzol in jede Vertiefung und das Bestehen der Zelllysat mehrmals in eine Pipette. Die homogenisierte Proben inkubieren 5 bis 10 Minuten bei Raumtemperatur, um die vollständige Dissoziation Nukleoproteinkomplexen ermöglichen. </li><li> Übertragen Lysat in 1,5 ml RNase- und DNase-freie Mikrozentrifugenröhrchen. 0,2 ml Chloroform pro 1 ml TRIzol. Durch kräftiges Schütteln Rohre von Hand für 15 Sekunden und inkubieren bei Raumtemperatur für 5 bis 10 min. Zentrifuge bei 12.000 xg für 15 min bei 4 ° C. Beachten Sie die Mischung getrennt wird in eine untere rote Phase (Phenol-Chloroform), eine Interphase und eine farblose obere wässrige Phase. RNA liegt ausschließlich in der wässrigen Phase. </li><li> Transfer sorgfältig die obere wässrige Phase in ein frisches Röhrchen, ohne die Interphase zu stören. Ausfällung der RNA aus es durch Mischen mit 0,5 ml Isopropylalkohol. Proben Inkubation bei RT für 10 Minuten und Zentrifugation bei 12.000 × g für 10 min bei 4 ° C. Die RNA-Ausscheidungen bildet ein weißes Pellet an der Seite und am Boden des Röhrchens. </li><li> Überstand entfernen. Mit 1 ml 75% Ethanol waschen das RNA-Pellet einmal. Mischen Sie die Proben durch Vortexen und Zentrifugieren bei 7.500 xg für 5 Minuten bei 4 ° C. Kurz trocknen die RNA (Luft trocknen für 10 min). Lösen sich RNA in 0,1 ml DEPC (RNAse freies Wasser) und Inkubation für 10 min bei 55 ° C. </li><li> Quantifizieren RNA mit einem Spektralphotometer. Um dies zu tun, verdünnte Proben 100 Mal in DEPC Wasser und bei Wellenlängen von 260 nm gelesen. Die RNA-Konzentration ist dann: OD 260 x 40 ng / ul x Verdünnungsfaktor. </li><li> RNA-Konzentrationen auszugleichen und zu synthetisieren cDNA gemäß den Anweisungen des Herstellers unter Verwendung von RT-PCR-System für First-Strand cDNA Synthesis Kit. Wieden Anweisungen des Herstellers, messen Gen-mRNA-Spiegel durch Echtzeit-PCR (45 Zyklen) in einer Echtzeit-DNA-Detektionssystem. Normalisierung der Genexpression Ebenen mit zwei Housekeeping-Gen beispielsweise β-Actin und GAPDH. HINWEIS: Normalisieren RNA-Konzentration auf 500 ug / ml und mit 0,5 &amp; mgr; g für die cDNA-Synthese. </li></ol>

<ol>
	<li>Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+macrophages+in+development+and+disease.">Trophic macrophages in development and disease.</a> Nat Rev Immunol. 9 (4), 259-270 (2009).</li><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 (1), 23-35 (2003).</li><li>Koury, M. J., Ponka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+erythropoiesis:+the+roles+of+folate,+vitamin+B12,+and+iron.">New insights into erythropoiesis: the roles of folate, vitamin B12, and iron.</a> Annu Rev Nutr. 24, 105-131 (2004).</li><li>Sheftel, A. D., Kim, S. F., Ponka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-heme+induction+of+heme+oxygenase-1+does+not+alter+cellular+iron+metabolism.">Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism.</a> J Biol Chem. 282 (14), 10480-10486 (2007).</li><li>Hershko, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+iron+regulation.">Storage iron regulation.</a> Prog Hematol. 10, 105-148 (1977).</li><li>Collins, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Withholding+iron+as+a+cellular+defence+mechanism--friend+or+foe.">Withholding iron as a cellular defence mechanism--friend or foe.</a> Eur J Immunol. 38 (7), 1803-1806 (2008).</li><li>Noyes, W. D., Bothwell, T. H., Finch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+reticulo-endothelial+cell+in+iron+metabolism.">The role of the reticulo-endothelial cell in iron metabolism.</a> Br J Haematol. 6, 43-55 (1960).</li><li>Layoun, A., Huang, H., Calve, A., Santos, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signal+adaptor+protein+MyD88+is+required+for+sustained+endotoxin-induced+acute+hypoferremic+response+in+mice.">Toll-like receptor signal adaptor protein MyD88 is required for sustained endotoxin-induced acute hypoferremic response in mice.</a> Am J Pathol. 180 (6), 2340-2350 (2012).</li><li>Zumerle, S., 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=Targeted+disruption+of+hepcidin+in+the+liver+recapitulates+the+hemochromatotic+phenotype.">Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype.</a> Blood. 123 (23), 3646-3650 (2014).</li><li>Nguyen, N. B., Callaghan, K. D., Ghio, A. J., Haile, D. J., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepcidin+expression+and+iron+transport+in+alveolar+macrophages.">Hepcidin expression and iron transport in alveolar macrophages.</a> Am J Physiol Lung Cell Mol Physiol. 291 (3), L417-L425 (2006).</li><li>Nemeth, E., 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=Hepcidin,+a+putative+mediator+of+anemia+of+inflammation,+is+a+type+II+acute-phase+protein.">Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.</a> Blood. 101 (7), 2461-2463 (2003).</li><li>Nemeth, E., 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=Hepcidin+regulates+cellular+iron+efflux+by+binding+to+ferroportin+and+inducing+its+internalization.">Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.</a> Science. 306 (5704), 2090-2093 (2004).</li><li>Constante, M., 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=Distinct+requirements+for+Hfe+in+basal+and+induced+hepcidin+levels+in+iron+overload+and+inflammation.">Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation.</a> Am J Physiol Gastrointest Liver Physiol. 291 (2), G229-G237 (2006).</li><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Curr Protoc Immunol. Chapter 14 (Unit 14 11), (2008).</li><li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+peritoneal+cavity+cells.">Isolation of mouse peritoneal cavity cells.</a> J Vis Exp. (35), (2010).</li><li>Leijh, P. C., van Zwet, T. L., ter Kuile, M. N., van Furth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thioglycolate+on+phagocytic+and+microbicidal+activities+of+peritoneal+macrophages.">Effect of thioglycolate on phagocytic and microbicidal activities of peritoneal macrophages.</a> Infect Immun. 46 (2), 448-452 (1984).</li><li>Dy, M., Debray-Sachs, M., Kamoun, P., Hamburger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thioglycollate+on+macrophage+lysosomal+enzymes.">Effect of thioglycollate on macrophage lysosomal enzymes.</a> Biomedicine. 29 (5), 167-170 (1978).</li><li>Li, Y. M., Baviello, G., Vlassara, H., Mitsuhashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+products+in+aged+thioglycollate+medium+enhance+the+elicitation+of+peritoneal+macrophages.">Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages.</a> J Immunol Methods. 201 (2), 183-188 (1997).</li><li>Layoun, A., Santos, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cell+wall+constituents+induce+hepcidin+expression+in+macrophages+through+MyD88+signaling.">Bacterial cell wall constituents induce hepcidin expression in macrophages through MyD88 signaling.</a> Inflammation. 35 (4), 1500-1506 (2012).</li><li>Oppenheim, J. J., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alarmins:+chemotactic+activators+of+immune+responses.">Alarmins: chemotactic activators of immune responses.</a> Curr Opin Immunol. 17 (4), 359-365 (2005).</li><li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124 (4), 783-801 (2006).</li><li>Lang, R., Patel, D., Morris, J. J., Rutschman, R. L., Murray, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+gene+expression+in+activated+and+resting+primary+macrophages+by+IL-10.">Shaping gene expression in activated and resting primary macrophages by IL-10.</a> J Immunol. 169 (5), 2253-2263 (2002).</li><li>Wang, C., 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=Characterization+of+murine+macrophages+from+bone+marrow,+spleen+and+peritoneum.">Characterization of murine macrophages from bone marrow, spleen and peritoneum.</a> BMC Immunol. 14 (6), (2013).</li><li>Austin, P. E., McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture.">Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture.</a> J Cell Physiol. 77 (2), 121-134 (1971).</li><li>Cannon, G. J., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+capacity+for+phagocytosis.">The macrophage capacity for phagocytosis.</a> J Cell Sci. 101 (Pt 4), 907-913 (1992).</li><li>Wang, Y., Harris, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+renal+disease.">Macrophages in renal disease.</a> J Am Soc Nephrol. 22 (1), 21-27 (2011).</li><li>Edwards, J. P., Zhang, X., Frauwirth, K. A., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+functional+characterization+of+three+activated+macrophage+populations.">Biochemical and functional characterization of three activated macrophage populations.</a> J Leukoc Biol. 80 (6), 1298-1307 (2006).</li><li>Hoover, D. L., Nacy, C. A. <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+to+kill+Leishmania+tropica:+defective+intracellular+killing+of+amastigotes+by+macrophages+elicited+with+sterile+inflammatory+agents.">Macrophage activation to kill Leishmania tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents.</a> J Immunol. 132 (3), 1487-1493 (1984).</li><li>Schleicher, U., Bogdan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+culture+and+flow-cytometric+characterization+of+primary+mouse+macrophages.">Generation culture and flow-cytometric characterization of primary mouse macrophages.</a> Methods Mol Biol. 531, 203-224 (2009).</li></ol>

The authors have no competing financial interests.

Makrophagen sind von entscheidender Bedeutung für das Überleben und ein verlockendes Ziel, um den Host für immunologische Ziele zu manipulieren. Die Entdeckung von TLR und andere Erkennungsmoleküle das Makrophagen zur Mitte der immunologischen Debatte geführt. Makrophagen sind auf eine Vielzahl von Stimuli, einschließlich Cytokinen, Schadens-associated molecular pattern Moleküle (dämpft) 20 und Moleküle mit Gruppen von Pathogenen (PAMPS) 21 verbunden. Diese Reaktionen unterschiedliche Stim...

Retikuloendothelialen phagozytische System von Zellen in verschiedenen Geweben und Organen, wie Knochenmark, Blut, Leber und Milz besteht. Makrophagen werden in großem Umfang um den Körper, wo sie vor allem in angeborenen teilzunehmen und adaptiven Immunantwort zu steuern und zu Infektionen klar verteilt. Zusätzlich zu ihrer Rolle bei der Immunabwehr, Makrophagen spielen eine wichtige Rolle bei der Wundheilung und bei der Aufrechterhaltung Gewebshomöostase 1,2. Darüber hinaus sind Makrophagen nicht nur die Immunfunktion wichtig, sondern auch aktiv in Eisenhomöostase 3 teilzunehmen. Im Körper ist etwa 80% der Eisen im Hämoglobin in Erythrozyten vorhanden ist, die bei seneszenten werden von Makrophagen phagozytiert 4. Täglich, diese Makrophagen recyceln 25 mg von Erythrozyten-abgeleitete Eisen und bringt ihren Transport in das Plasma 5. Darüber hinaus während der Infektion und Entzündung, proinflammatorischen Makrophagen maskieren Serum-Eisen auf Eisen VERFÜGBARK reduzierenty, um Krankheitserreger, die an beiden systemischen und lokalen Ebenen 6-8. Wie gut, Studien haben gezeigt, dass vor allem Makrophagen und Hepatozyten erzeugen ein antimikrobielles Peptid namens Hepcidin, das als das Master-Regulator des Eisenstoffwechsels 9, 10 ist. Hepcidin wird hauptsächlich durch inflammatorische Stimuli erhöht und ist teilweise für die Eisenspeicherung in Makrophagen bei chronischen Entzündungen 11-13 verantwortlich. Wie Hepcidin-Expression in Makrophagen ist nicht sehr gut verstanden, haben wir untersucht die mögliche Rolle von Toll-like Rezeptoren (TLRs) in dieser Regelung. Die TLRs sind in erster Linie auf Makrophagen und spielen eine zentrale Rolle in der Aktivierung. Zusätzlich ist LPS induzierte Hepcidin Expression in der Leber hängt von TLR4 13. Daher unserer Untersuchung auszuführen, verwendeten wir ein Verfahren, das auf die Isolierung von murinen peritonealen Makrophagen. Makrophagen-Zelllinien werden im Großen und Ganzen in Makrophagen Zucht eingesetzter Jahren; dennoch ausgedehnte Kultur Genverluste provozieren und verringerte Immunfunktionen in diesen Zelllinien. Somit ist die Isolierung von Makrophagen aus der Peritonealhöhle entscheidend. Die Maus Bauchhöhle stellt eine ideale Ort, um Makrophagen 13-15 ernten. Isolated murinen peritonealen Makrophagen sind bequem für mehrere Studien in Bezug auf ihre immunologische Funktion. Jedoch ist die Anzahl von Makrophagen in das Peritoneum nicht ausreichend ist für umfangreiche Untersuchungen und wird geschätzt etwa 1 x 10 6 Makrophagen pro Maus. Somit Makrophagen Ausgang zu erhöhen, wurde eine sterile Hervorrufen Mittel wie Thioglykolat in die Bauchhöhle vor der Zellernte injiziert. Nach Thioglycolat Injektion wurde die Ausbeute an Makrophagen pro Maus durch das 10-fache erhöht. Trotz der Zunahme der Makrophagen Ertrag, Thioglycolat-Medium wirkt Brewer als Reizmittel, die eine Entzündungsreaktion induziert, was zur Rekrutierung von Makrophagen, die may, aber nicht notwendig Auswirkungen auf die Genexpression. Daher muss eine Kontrollgruppe, bestehend aus nicht-behandelten Makrophagen in jedem Experiment eingeschlossen sein. In unseren Händen, wurde Hepcidin-Expression, die stark durch eine Entzündung stimuliert wird nicht in nicht-behandelten Thioglycolat erkannt ausgelöst Peritonealmakrophagen. Darüber hinaus haben Studien gezeigt, dass Brewer Thioglycolat rekrutiert zahlreiche Makrophagen, aber nicht 16 aktivieren. Andererseits löste Brewer Thioglycolat Makrophagen zeigten einen Anstieg der lysosomalen Enzyms, aber eine Verringerung in der Tötung von Mikroorganismen 17 eingenommen. Allerdings wurde die Phagozytose nicht, wenn sie mit nicht-ausgelöst Makrophagen 16 im Vergleich betroffen. Einmal in Schalen kultiviert, die Peritonealmakrophagen verwachsen und ermöglicht damit ihre Trennung von einer anderen Art von Zellen, die aus der Peritonealhöhle isoliert wird. Anschließend wurden die isolierten Makrophagen mit verschiedenen TLR Agonisten herausgefordert.Schließlich mRNA wurde aus den kultivierten Zellen extrahiert, und die Genexpression wurde durch quantitative reverse Transkriptase-Polymerasekettenreaktion (qRT-PCR) analysiert.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td>
			<td>475</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycollate</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>19032-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td>
			<td>WISENT INC Canada (QC)</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td>
			<td>WISENT INC Canada (QC)</td>
			<td>350-000-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 and 5 ml syringes</td>
			<td>BD USA (NJ)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>20g and 23g needles</td>
			<td>BD USA (NJ)</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes placed on ice</td>
			<td>Sarstedt (Newton, MA, USA)</td>
			<td>62.547.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cells Lysis Buffer</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>R7757-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80 antibody</td>
			<td>BIO-RAD ( CA, USA)</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32 antibodies</td>
			<td>Pharmingen, {Mississauga, ON, CA)</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>Axygen Scietific (CA,USA)</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific (ON, Canada)</td>
			<td>UN1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>JT Baker (PA, USA)</td>
			<td>70566</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol (in DEPC treated water)</td>
			<td>Commercial Alchohols (QC, Canada)</td>
			<td>17394</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>216.542.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript RT-PCR system</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor Gene 3000</td>
			<td>Montreal Biotech, (Kirkland, QC, Canada)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect SYBR Green I PCR kits</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>204141</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

Während der Infektion und Entzündung, lassen zirkulierenden Monozyten in die Blutbahn und wandern ins Gewebe, wo sie in Makrophagen zu differenzieren. Makrophagen exprimieren Oberflächen Toll-like Rezeptoren (TLRs), die molekulare Muster in einer Vielzahl von Mikroorganismen durch Evolution konserviert zu erkennen. TLRs spielen eine zentrale Rolle in Makrophagenaktivierung, die in der Regel mit Genexpressionsänderung verbunden ist. Makrophagen sind kritisch bei vielen Krankheiten und werden als attraktive Ziele für eine Therapie entwickelt. In dem folgenden Protokoll beschreiben wir ein Verfahren, um murine peritoneale Makrophagen mit Brauerei Thioglykolat-Medium zu isolieren. Letzteres wird Monozytenmigration in das Peritoneum zu steigern, entsprechend wird dies Makrophagen-Ausbeute um das 10-fache zu erhöhen. Mehrere Studien wurden unter Verwendung von Knochenmark, Milz und peritoneale Makrophagen durchgeführt. Allerdings wurden peritoneale Makrophagen gezeigt reiferen auf Isolation zu sein und sind in ihrer funktionalen stabilerkeit und Phänotyp. So Makrophagen aus murinen Bauchhöhle isoliert präsentieren eine wichtige Zellpopulation, die in verschiedenen immunologischen und metabolischen Untersuchungen dienen kann. Nach der Isolierung wurden Makrophagen mit verschiedenen TLR-Liganden stimuliert und damit die Genexpression wurde ausgewertet.

Zunächst gekennzeichnet die isolierten murinen peritonealen Makrophagen mittels Durchflusszytometrie. Um dies zu tun, haben wir (F4 / 80) Antikörper, die spezifisch Marker nur durch Makrophagen zu erkennen. Diese Charakterisierung wird benötigt, um die Prozentzahl der isolierten Makrophagen zu bestimmen und sie unter den Zellen während des Isolierungsprozesses erhaltenen unterscheiden. Wie in (1) gezeigt, ist der Prozentsatz von Zellen, die das Antigen F4 / 80 wurde übereinstimmend festgestellt, ü...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolierung von Murine Peritonealmakrophagen zur Durchführung Genexpressionsanalyse Nach Toll-like Rezeptoren Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.0K Aufrufe.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Serie: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Isolierung von Maus Bauchhöhle Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Forschung

Immunologie und Infektion

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Erkennung von Neu1 Sialidase-Aktivität bei der Regulierung der Toll-like-Rezeptor-Aktivierung

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Quantitative Bildgebung von Lineage-spezifischen Toll-like Rezeptor-vermittelten Signaltransduktion in Monozyten und dendritischen Zellen aus kleinen Proben des menschlichen Blutes

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolation, Verarbeitung und Analyse von Murine Gingivazellen

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Verwendung von<em&gt; Shigella flexneri</em&gt; Zum Autophagie-Zytoskelett Interaktionen zu untersuchen

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

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila der äußeren Membran - Vesikeln: Isolierung und Analyse ihrer Pro-inflammatorische Potential auf Makrophagen

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolierung von Salmonella Typhimurium-mit Phagosom von Makrophagen

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

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Mit der phylogenetischen Analyse zu eukaryotischen Gens Herkunft untersuchen

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Bewertung der Antikörper-basierte Medikamente Auswirkungen auf murinen Knochenmark und peritonealen Makrophagen Aktivierung

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

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