Bu eser Doğa Bilimleri ve Kanada&#39;nın Mühendislik Araştırma Konseyi bir hibe ile desteklenmiştir (NSERC, hayır 298515-2011 hibe). AL doktora almıştır Doğa Bilimleri ve Kanada&#39;nın (NSERC) Mühendislik Araştırma Konseyi ve MS burs (hayır verin. MOP123246 CIHR), Kanada Sağlık Araştırma Enstitüleri bir hibe desteklenmiştir.

Tüm işlemler Centre de recherche du Centre Hospitalier de l&#39;Université de Montréal kurumsal Hayvan Bakım Komitesi (CRCHUM) tarafından onaylandıktan sonra Hayvan Bakımı yönergelere Kanada Konseyi&#39;ne göre yapıldı. 1. İzolasyon, Kimlik ve Kültür Kemirgen Periton Makrofagların <ol><li> % 3.8 bira tiyoglikolat orta hazırlayın. Bunu yapmak için, damıtılmış su içinde 1000 ml tiyoglikolat vasatı içinde 38 g askıya almak için. Tamamen orta çözmek için kaynamaya çözüm getirin. 15 dakika boyunca 121 ° C&#39;de otoklav ile sterilize edin. RT 18 at, karanlıkta 3 aya kadar saklayabilirsiniz. NOT: Bulanıklık bakteriyel kontaminasyon işaret eden gelişirse atın. Steril tutulması durumunda çözüm karanlıkta bir yıla kadar muhafaza edilebilir. </li><li> Her MZ&#39;nin periton boşluğu içine% 3.8 Brewer, tiyoglikolat vasatı 1 ml enjekte edilir, 23 G iğne takılı 1 ml şırınga kullanılarakse 3 gün bekleyin ve. Her fare için yeni şırınga ve iğne kullanın. </li><li> Sodyum pentobarbital (100 mg / kg) intraperitonal enjeksiyonu ile fareler anestezi ve servikal dislokasyon ile farelerin euthanize. Solunum hızı kontrol ederek uygun anesthetization onaylayın. Genellikle hızlı Solunum fare derin anestezi yapılmadan olduğunu göstermektedir. </li><li> % 70 etanol ile her bir farenin karın yıkayın. Bir makas kullanarak, periton alt orta hat boyunca bir yanal kesi yapmak. </li><li> Bir forseps kullanarak, şeffaf periton cilt ortaya çıkarmak için karın cilt geri çekin. 20 G iğne takılı 5 mi şırınga kullanarak, her bir farenin karın boşluğu içine soğuk DPBS 5 ml enjekte edilir. </li><li> Periton boşluğuna üzerinde yumuşak bir masaj yapın ve sonra herhangi bir organı delinmesiyle olmadan dikkatlice sıvıyı aspire. İğneyi çıkarın ve 50 ml konik santrifüj tüplerine periton sıvısı dağıtmak. </li><li> Soğutuculu santrifüj 400 x g&#39;de 10 dakika boyunca santrifüj.Hücreler tüm prosedür sırasında soğuk kalmalıdır. RPMI ortamı 1640 süpernatant ve tekrar süspansiyon hücre pelletini atın. </li><li> Bir hemasitometre kullanarak, hücrelerin sayısı ve 1 x 10 6 hücre / ml hücre yoğunluğuna kadar ayarlayın. </li><li> F4 / 80 karşı fare ve Antikorların başına 1 x 10 6 hücreleri kullanılarak akış sitometrisi ile izole edilmiş hücrelerin fenotipini karakterize (bir yüzey antijeni makrofajlar üzerinde ifade edilir). </li></ol> 2. Hücre Tedavileri <ol><li> Doğrudan, izolasyondan sonra, her kuyuya 1 x 10 6 hücre ekleyin. 37 ° C&#39;de 1 saat ila 2 içinde kültürlenmesi ile 6-çukurlu plakalara yapışmaya sıçan periton makrofajları, bırakın. Hafifçe sıcak PBS ile 3 kez yıkanarak yapışmayan hücreler çıkarın. </li><li> Daha sonra, 900 ul serum içermeyen DMEM içinde kültür hücreleri, şu TLR ligandlar varlığında, 24 saat süre ile (Pam3CSK4-TLR1 / 2 (0.5 mg / ml); Poli (I: C) -TLR3 (10 mg / ml) LPS-TLR4 (100 ng / ml); flagellin&#39;e TLR5 (100 ng / ml); FSL1-TLR6 / 2 (100 ng / ml); ssRNA40-TLR7 (1 ug / ml); ODN1826-TLR9 (1 uM). NOT: Her ligand 10x stok solüsyonu için hazırlayın. Bu şekilde, bir 10-kat seyreltme yapılarak, her bir ikincisi, 100 ul ekle. </li></ol> 3. RNA İzolasyon <ol><li> Bir pipet ile birkaç kez her kuyuya, 1 ml Trizol eklenmesi ve hücre lizatı geçen altı oyuklu plakalar içinde doğrudan tüm orta ve lize hücreleri çıkarın. Nükleoprotein kompleksinin tam bir ayrışma izin vermek için, oda sıcaklığında 5-10 dakika boyunca homojenize örnekleri inkübe edin. </li><li> 1.5 ml lizat transfer RNase- ve DNaz içermeyen mikrosantrifüj tüpleri. 1ml TRIzol başına 0.2 ml kloroform ekleyin. 15 saniye boyunca elle kuvvetli bir şekilde tüpleri çalkalanır ve 5-10 dakika boyunca oda sıcaklığında inkübe. 4 ° C&#39;de 15 dakika boyunca 12,000 x g&#39;de santrifüjleyin. Bir düşük değerli kırmızı renkte bir faz (fenol-kloroform) ayrılarak karışımı, bir faz ve renksiz bir üst sulu faz dikkate alınmalıdır. RNA, sulu faz içinde özel olarak kalır. </li><li> Traninterfazda bozmadan, yeni bir tüpe Sfer dikkatle üst akıcı faz. Izopropil alkol, 0.5 ml ile karıştırılarak bu RNA hızlandırabilir. 4 ° C&#39;de 10 dakika boyunca 12,000 x g&#39;de 10 dakika santrifüj boyunca oda sıcaklığında inkübe edin. RNA tüpün yan ve alt beyaz pelet oluşturan çökeltileri. </li><li> Süpernatantı. % 75 etanol içinde bir kez 1 mL RNA pelet yıkayın. 4 ° C&#39;de 5 dakika boyunca 7500 x g&#39;de vorteks ve santrifüj ile örnekleri karıştırın. Kısaca RNA (10 dakika boyunca hava kuru) kurutun. 0.1 mi DEPC (RNaz içermeyen su) içinde RNA eritin ve 55 ° C&#39;de 10 dakika inkübe edilir. </li><li> Bir spektrofotometre kullanılarak RNA niceliğini. Bunu yapmak için numuneler DEPC su içinde 100 kez seyreltilmesi ve 260 nm dalga boyunda okundu. RNA konsantrasyonu daha sonra olacak: OD 260 x 40 ng / ul x seyreltme faktörü. </li><li> RNA konsantrasyonları eşitlemek ve İlk Strand cDNA Sentezi Kit RT-PCR Sistemi kullanılarak üreticinin talimatlarına göre cDNA sentez. Olaraküreticinin talimatlarına göre gerçek zamanlı PCR, bir gerçek zamanlı DNA tespit sisteminde (45 devir) ile gen mRNA seviyelerini ölçer. Örnek β-aktin ve GAPDH için iki temizlik geni ile gen ekspresyon düzeyleri Normale. NOT: ml 500 ug / RNA konsantrasyonu Normale ve cDNA sentezi için 0.5 ug kullanın. </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.

Makrofajlar hayatta kalmak için çok önemlidir ve immünolojik hedefler için ana işlemek için cazip bir hedef sağlar. TLR ve diğer tanıma moleküllerinin keşfi immünolojik tartışmaların merkezine makrofajlar yaptık. Makrofajlar sitokinler, hasar ile ilişkili molekül model molekülü (DAMPS), 20 ve patojenlerin (PAMPs) 21 grupları ile ilişkili moleküller de dahil olmak üzere çeşitli uyaranlara, yanıt. Bu farklı uyaranlara yanıt makrofaj aktivasyonu seyrini temsil etmektedir ...

retiküloendotelyal fagositik sistemi, kemik iliği, kan, karaciğer ve dalak gibi çeşitli dokularda ve organlarda hücrelerden oluşmaktadır. Makrofajlar yoğun bunlar, özellikle doğuştan katılmak ve kontrol bağışıklık tepkilerini ve net enfeksiyonları uyarlamalı vücudun etrafında dağıtılır. Host savunmasında rollerine ilave olarak, makrofajlar, yara iyileşmesi ve doku homeostazını 1,2 muhafaza edilmesinde önemli bir rol oynamaktadır. Ayrıca, makrofajlar bağışıklık işlevi sadece önemli değil, aynı zamanda aktif demir homeostazda 3 katılacak. Vücutta demir yaklaşık% 80&#39;i yaşlanmış makrofajlar tarafından fagosite 4 hangi zaman, eritrositler içinde hemoglobin bulunur. Günlük, bu makrofajlar eritrosit kaynaklı demir 25 mg geri dönüşüm ve plazma 5 içine ulaşım sağlamaktadır. Ayrıca, enfeksiyon ve enflamasyon sırasında, pro-inflamatuar makrofajlar demir availabili azaltmak için serum demir çekilmekhem sistemik ve yerel düzeylerde 6-8 patojenlerin için ty. Makrofajlar ve özellikle hepatositler demir metabolizmasının 9, 10 master regülatörü olarak kabul edilir hepsidin adında bir antimikrobiyal peptid üretmek Yanı sıra, çalışmalar göstermiştir. Hepsidin esas inflamatuar uyaranlara artmış ve kronik inflamasyon 11-13 üzerine makrofaj demir haciz kısmen sorumludur. Makrofajlarda hepcidin ifadesi çok iyi anlaşılamamıştır, biz bu yönetmelikte Toll benzeri reseptörler olası rolünü (TLR) okudu. TLR&#39;ler öncelikle makrofajlar üzerinde bulunan ve aktivasyon merkezi bir rol oynarlar. Buna ek olarak, karaciğer LPS kaynaklı Hepsidin sentezleme TLR4 13 bağlıdır. Bu nedenle, çalışma çalıştırmak için, Sıçan peritonal makrofajları izolasyonu dayanan bir yöntem kullanılır. Makrofaj hücre dizileri genel olarak, makrofaj saplama kullanılanler; yine genişletilmiş kültür geni kaybını kışkırtmak ve bu hücre hatlarında bağışıklık fonksiyonları azalmış olabilir. Bu nedenle, periton boşluğundan makrofajların izolasyonu önemlidir. Fare periton boşluğu makrofajlar 13-15 hasat için ideal bir siteyi sunuyor. İzole fare periton makrofajlar onların immünolojik fonksiyonu ile ilgili çeşitli çalışmalar için uygundur. Ancak, periton makrofajların sayısı geniş çalışmalar için yetersiz ve fare başına yaklaşık 1 x 10 6 makrofajlar tahmin edilmektedir. Bu nedenle, makrofaj üretimini artırmak için, örneğin tiyoglikolat gibi steril ortaya çıkarma maddesi, hücre hasat Yukarıdaki periton boşluğu içine enjekte edilmiştir. Tiyoglikolat enjeksiyonundan sonra, fare başına makrofajların verimi 10-kat artmıştır. Makrofajlar verim, makrofajlar alımı, MA içinde elde edilen, bir enflamatuvar yanıtı indükleyen bir tahriş edici olarak Brewer, tiyoglikolat vasatı eylemleri artışa rağmeny, ancak gerekli değildir, gen ekspresyonunu etkiler. Bu nedenle, muamele edilmemiş makrofaj oluşan bir kontrol grubudur, her bir deney içinde dahil edilmelidir. Bizim ellerde, çok inflamasyon ile uyarılır hepsidin ifade periton makrofajlar ortaya dışı muamele tiyoglikolatın saptanmadı. Ayrıca, çalışmaların Bira tiyoglikolat sayıda makrofajların, fakat bunlara 16 aktive etmediğini göstermiştir. Öte yandan, Bira tiyoglikolat makrofajlar lizozomal enzim bir artış ancak yutulur mikroorganizmaları öldürme 17 azalma gösterdi ortaya çıkardı. Olmayan tetiklenen makrofajlar 16 ile karşılaştırıldığında, ancak, fagositik kapasitesi etkilenmedi. Yemekler kültürlenen sonra peritoneal makrofajlar nedenle periton boşluğu izole diğer hücre türünden bunların ayrılmasına izin veren, yapışkan hale gelir. Daha sonra izole edilmiş makrofajlar, farklı TLR agonistleri ile tehdit edildi.Son olarak, mRNA, kültürlenmiş hücrelerden ekstrakte edildi ve gen ekspresyonu kantitatif ters transkriptaz-polimeraz zincir reaksiyonu (qRT-PCR) kullanılarak analiz edilmiştir.

<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

Enfeksiyon ve inflamasyon sırasında dolaşımdaki monositler kan dolaşımına bırakın ve onlar makrofajlar içine ayırt dokulara, göç. Makrofajlar mikroorganizmaların geniş bir yelpazede evrimle korunmuş moleküler şekilleri tanıyan yüzey Toll-benzeri reseptörler (TLR), ifade eder. TLR genellikle gen ekspresyonu değiştirilmesi ile ilişkili makrofaj aktivasyonunda önemli bir rol oynar. Makrofajlar pek çok hastalıkta önemli ve tedavisi için çekici bir hedef olarak ortaya çıkmıştır. Aşağıdaki protokol, bira, tiyoglikolat vasatı kullanılarak sıçan periton makrofajları, izole edilmesi için bir prosedür açıklanmaktadır. İkincisi buna göre bu 10 kat makrofaj verim çıkaracağız, periton içine monosit göçü artıracak. Pek çok çalışma, kemik iliği, dalak veya periton türetilmiş makrofajlar kullanılarak gerçekleştirilmiştir. Ancak, periton makrofajlar izolasyon üzerine daha olgun olduğu gösterilmiştir ve bunların fonksiyonel olarak daha kararlı edildiSığ ve fenotip. Böylece, fare periton boşluğuna izole makrofajlar farklı immünolojik ve metabolik çalışmalarda hizmet verebilir önemli bir hücre popülasyonu sunuyoruz. İzole bir kez, makrofajlar farklı TLR ligandları ile uyarıldı ve dolayısıyla gen ifadesi değerlendirildi.

İlk akış sitometrisi ile izole edilmiş sıçan periton makrofajları, karakterize edilir. Bunu yapmak için, özellikle yalnızca makrofajlar tarafından ifade edilen markerler tanır (F4 / 80) antikorları kullanılmıştır. Bu karakterizasyon izole makrofaj yüzdesini belirlemek için ve izolasyon işlemi sırasında elde edilen hücreler arasında onları ayırmak için gereklidir. (Şekil 1) gösterildiği gibi, hücrelerin yüzdesi F4 / 80 sürekli olarak% 95 üzerinde olduğu tespit edilmişt...

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.

Fare Periton Makrofagların izolasyonu Toll-benzeri reseptörler Uyarım üzerine Gen İfadesi Analizi Yapacak

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 Görüntüleme.  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.

Video: 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.

Fare Periton Boşluk Hücreleri İzolasyon

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

İmmünoloji ve Enfeksiyon

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.

Toll-like reseptör Etkinleştirme Düzenlenmesi Neu1 Sialidase Faaliyet Algılama

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.

Monositler ve İnsan Kan Küçük Örneklerden Dendritik Hücrelerde Lineage özgü Toll-like reseptör-aracılı Sinyali Kantitatif Görüntüleme

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.

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

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.

Kullanımı<em&gt; Shigella flexneri</em&gt; Otofaji-Sitoiskelet Etkileşimleri Okumak

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 Dış Zar veziküller: İzolasyon ve Makrofajlar Onların Pro-inflamatuar Potansiyeli Analizi

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.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

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.

Ökaryotik gen kökenli araştırmaya filogenetik analizi kullanılarak

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.

Fare kemik iliği ve Periton makrofaj harekete geçirmek antikor bazlı ilaçlara etkileri değerlendirilmesi

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.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

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