Bu çalışma NIEHS R01ES014470 (KMZ) tarafından desteklenmiştir.

Önce bu protokolü başlayan, doğum sonrası günlerde yenidoğan farelerin toplamak 1-3 koruma ve sıcaklık için özgün kafes yatak ile iç içe bir steril kap içinde (P1-3). Bu mikrogliyal verimi optimize etmek, bu protokol sayesinde hızlı ve verimli bir şekilde çalışmak önemlidir. Tam reaktif listesi için Tablo 1&#39;e bakınız lütfen. 1.. Aletleri, Kültür, Medya ve Yemekleri hazırlanması <ol><li> 10 ml / pup Mikroglia tam ortam maddesi (MCM hazırlanması; 1x Minimal Essential ile desteklenmiş Medium Earle [MEM]: 1 mM L-glutamin, 1 mM sodyum piruvat,% 0.6 v / v D-(+)-glukoz, 100 ug / ml Penisilin / Streptomisin (P / S),% 4 h / h cenin sığır serumu (FBS), 6 v / v% at serumu) ve her bir T-75 balonuna (1 şişe / yavru) eklemek, kapak ve yeri yatay olarak bir inkübatör (37 ° C,% 5 CO2), 1 saat boyunca dengelenmeye. O diseksiyon başlamadan önce bir şişe (ler) de bu ortamı dengeye için gereklidir. </li><li> Prepare, kısım ve sıcak diseksiyon (6 ml / pup) için 37 ° C su banyosu MCM ve hücre yeniden süspansiyon (2 mi / yavru) için ayrı bir konik tüp MCM halinde. Ve Kesme Media (DM; MEM 100 ug / ml ile takviye edilmiş P /: buz Kıyma Media (,, 100 ug / ml P / S, 0.01 M HEPES 3 ml / pup HBSS ile takviye MM) hakkında Hazırlama, kısım ve soğuk S, 2 ml / pup). </li><li> 10 dakika boyunca% 70 etanol içinde boğulma yolu ile aşağıdaki cerrahi aletleri sterilize: makas # 1 (1; başını kesmek için), # 1 forcep (2; burun güvenli ve korteks kaldırmak için), makas # 2 (1; nonangled; açmak için kafa derisi ,) makas # 3 (1 meninksler kaldırmak için,), forcep # 2 (1;,) kafatası kaldırmak için forcep # 4 (2; # 3 (1 forcep) derisini çıkarmak için beyin dokusunu inceltmek için). Steril bir pad üzerinde kurumaya bırakın. </li><li> % 70 etanol ile yatay hava akımı iş istasyonu ve diseksiyon mikroskop aşağı silin. </li><li> Başını yakalamak için 2 steril, 100 mm Petri kapları içine DM dağıtın (3 ml, 1/3 yavrular çanak) ve diseksiyonu (1 ml;Diseksiyon önce buz üzerinde soğutulmuş); / yavru 1 tabak. Beyin dokusu (; 1 tabak / yavru 1 ml) kıyma için 1 steril, 100 mm Petri kabı içine MM dağıtın. </li></ol> 2. Beyin Dokusu Diseksiyon <ol><li> Ötenazi öncesinde, hafifçe bir laboratuar dokusu kullanılarak% 70 etanol ile boyun ve yavru başını sterilize. Makas # 1, hızla başını kaldırarak yavru euthanize; yakalama kafasını önce hazırlanan Petri kabındaki. Euthanize ve sonraki hayvana geçmeden önce bir yavru diseksiyon tamamlayın. </li><li> Soğutulmuş diseksiyon çanak kafa transferi, açık kafa derisi bir oluşturarak makas # 2 kullanarak; diseksiyon mikroskobu altında # 1 (alternatif olarak, kafa göz soket aracılığıyla forseps piercing tarafından güvence altına alınabilir) forcep kullanarak burun sıkarak başını sağlamlaştırmak Y-cut: kafa tabanından ve gözleri doğru bir açıyla başlar. Yavaşça kafatası açığa yanal soyulması ile kafa derisi kaldırmak için 3. forcep kullanın. </li><li> Forcep 3. kullanarak, yavaşça tabanının en bağ dokusu kaldırmakkafa. Kavrama,, kafatası tabanı (interhemisferik fissür aşağıdaki) beyin ve kafatası arasında forcep bir kolunu dizilerek, dikkatli, açık kafatası maruz kaldığında yavaşça açık gözyaşı yukarı kafatası çekin. </li><li> Forcep # 1 kullanarak beynin ventral tarafında küçük bağ dokusu bozabilir ve daha sonra aynı aracı kullanarak beyin kaldırmak. Beynin ventral tarafında altında yukarı doğru bir kuvvet kullanılarak kafatası dışına beyin kaldırın. </li><li> Anatomik pozisyonda beyin tutun: sırt tarafında diseksiyon mikroskop altında korteks görselleştirmek için yukarı bakacak, orta beyinde ve korteks kesiştiği forseps ipuçlarını takarak yerine beyin sağlamak için; tamamen forcep # 4 ile kortikal meninksler kaldırmak, sonra ventral meninksler kaldırmak için devam edin. Zarları tamamen çıkarıldıktan sonra, forcep # 4 ile beyin kalan kısmından ayırmak korteks. Tamamen mikroglia saflık ve yiel üst düzeye çıkarmak için zarı doku kaldırmak için gereklidird. </li></ol> 3. Beyin Dokusu Kıyma <ol><li> Transferi Korteksler önce yemek kıyma hazırlanmış; Sınıf II Biyolojik Güvenlik Kabinesi steril tekniği kullanarak adımı kıyma devam ediyor. </li><li> Makas # 3 kullanılarak kıyma beyin dokusu, bir 15 ml konik tüp transfer kıyılmış doku,., Makas ve MM her 1 ml ile kıyma çanak durulayın toplamak ve kıyılmış doku içeren 15 ml konik tüp içine durulama dağıtmak Bu durulama adım maksimize edecek Tüm kalıntı doku konik tüpe aktarılır sağlayarak mikroglia verimi. Kıyılmış doku tüpün dibinde yerleşmek için izin, 1-2 dakika boyunca rahatsız doku süspansiyonu bırakın. MM gerekli besinleri içermez gibi 2 dk aşmayın. </li><li> Dikkatlice çıkarın ve yerleşmiş kıyılmış doku bozmak için değil bazı yapım, standart bir pipet ile jenerik 1.000 ul mikropipetöre ile 2ml&#39;si atın. </li&gt; <li> Kıyılmış-dokuya MCM 3 ml ekleyin, standart bir pipet ile bir jenerik 1.000 ul micropipettor kullanarak tam güçle doku çiğnemek trituration tamamlandığında hiçbir discernable katı doku kalmalıdır.. Pipet ucu kalıntı toz haline numune alınması, aynı pipet ucu ile MCM bir 3 ml ilave edilir. </li></ol> 4. Büyüyen Kortikal Hücreler <ol><li> Santrifüj hücreleri (215 xg, 5 dk; RT; sallanan bir kova rotor ile klinik santrifüj) pelet için kıyılmış doku. Biyolojik güvenlik kabini için pelet hücreleri dönün, kaldırmak ve süpernatant atılır; yavaşça hücre pelet (; MCM 2 ml / tüp); tekrar süspansiyon önceden hazırlanmış T-75 balona hücre tabanda ekleyin; şişesi kapağı ve içine yatay olarak yerleştirin , nemlendirilmiş, standart bir doku kültürü kuluçka makinesi (37 ° C,% 5 CO2). Hücreler, 24 saat boyunca inkübasyona izin verin. </li><li> 24 saat sonra, yavaşça bozmaya avucunuzda karşı şişeyi dokunundoku enkaz. Öncesi ve enkaz tümüyle kaldırılmasını sağlamak için dokunarak sonra hücrelerin kontrol edin. Set Şişe dikey, kaldırma ve ortam atılır; şişenin dibine yeni MCM ekle (12 mi, 37 ° C), kapağı ve yatay olarak geri inkübatör içine yerleştirin. </li><li> Kontaminasyon için günlük hücreleri kontrol ve büyümesini izlemek için. </li><li> In vitro (DIV) içinde yaklaşık 17-21 gün, Mikroglial hücreleri karışık glial kültür hasat için hazır olacak. Hücreler hazır olup olmadığını belirlemek için, bir ters faz-kontrast mikroskop altında hücrelerini inceleyen. Mikroglia hasat için hazır olduğunuzda% 40 confluency ~ at. Başka glia üstünde bir tek tabaka olarak büyüyen küçük, parlak, küresel hücreler olarak görünür. </li></ol> 5. Karışık Glial Kültür gelen mikrogliya İzolasyonu <ol><li> Mikroglianın ayırmak için, kuvvetli bir laboratuvar tezgah karşı şişesi dokunun. Bu ajitasyon mikroglia düşük yapışma özellikleri nedeniyle diğer glial hücrelerden ayrı mikroglia yardımcı olur. </li><li(; 37 ° C, 200 rpm, 5 saat). Parafilm ve karışık glial kültürlerin döndürmek&gt; ile Seal T-75 şişe kapakları sıcaklık kontrollü, nonhumidified döner karıştırıcı kullanılarak hücreler 5 saat daha uzun süre sallamak izin vermeyin gözle kontrol. bu mikroglia ters bir faz kontrast mikroskop kullanılarak, 5 saat sonra şişe yüzeyden kaldırdı. Mikroglia serbest yüzen hücreler, küre şeklinde, parlak. Astrositler ve şişenin dibine oligodendrosit yapışan bir kalıntı tabakası olacaktır. </li><li> Pellet mikroglia (215 x g,, 5 dak; kepçe rotor sallanan) süpernatantı steril bir konik santrifüj tüpüne mikroglia ihtiva eden ortam toplayın. </li><li> ~ 2,% 0.6 v / vglucose, 1 mM L-glutamin, 100 ug / ml P / S,% 5 v / v FBS 1 mM sodyum piruvat:; sıcak Mikroglial Büyüme Ortamı (MGM olarak mikroglial pelletini ile takviye edilmiş MEM ml / şişe), bir hemositometre kullanılarak, hücre konsantrasyonunu saptamak ve plaka 1 x 10, 24 oyuklu bir plaka biçiminde 5 hücre / gözplastik veya (500 ul / kuyu; MGM) steril cam kapak fişleri., bu prosedürü kullanarak ortalama mikrogliya verimi 7.5 x 10 5 hücre / yavru korteks. </li><li> 24-saat post-kaplama, yüzen hücreleri ve enkaz kaldırmak için taze MGM (37 ° C) ile medya ve tekrar çember yükleme hücreleri kaldırmak. Bu adım, hücrenin yaşamını ve sakin mikroglianın korumak için önemlidir. Hücreler hemen sonra refeeding deney için kullanılabilir. </li></ol> 6. Örnek Tedaviler <ol><li> Pam 3 CSK 4 (PAM) / ml, 10 ng / ml lipopolysaccharide (LPS) ve araç kontrolü 10 ng hücreleri açığa, nemlendirilmiş bir kuluçka makinesi içinde 2 ya da 24 saat süreyle inkübe edin (37 ° C,% 5 CO2). </li></ol> 7. İşlevsellik Ex vivo yoluyla Immunofluorescent Görüntüleme ve İmmünositokimya için mikrogliya Analizi <ol><li> Mikrofüj tüplerine (1.5 ml) ve centr tedavisi, hücre kültür ortamı hasat sonrasındamedyayı temizlemek için; ifuge bir masa mıkro (900 xg 2 dak) kullanarak. Temiz bir mikrofüj tüpüne süpernatant aktarın, tahlil derhal veya mağaza -20 ° C&#39;de kullanmaya hazır olana kadar. Ticari olarak temin edilebilen, fare TNF-α ELISA kiti ile hücre kültür tortusu içinde TNF-α konsantrasyonu analiz edin. </li><li> Fosfat tamponlu tuzlu su (PBS) hücreleri yıkayın ve% 4, hücreler (ağ / hac) paraformaldehit /% 4 (ağ / hac) sakroz / PBS pH 7.4 (% 4 PFA, 15 dakika, RT). Tespit sonra, yumuşak bir orbital çalkalayıcı üzerinde çalkalayarak 5 dakika boyunca PBS ile yıkama hücreleri. <ol><li> İmmünofloresan / İmmünositokimya. Sabitleme ve PBS immünsitokimya için, süreç mikroglianın yıkayın. Nazik sallayarak ile yürütülen tüm adımlar. PBS/0.1% triton X-100 hücreleri geçirgenleştirmek, bir saat boyunca PBS/10% normal keçi serumu içinde blok hücreler. Ya da bloke edici tampon içinde tavşan anti-Gözler-1 antikoru (1/750), tavşan anti-NF-KB antikoru (1/1, 250 p65 alt ünite) ile 4 ° C&#39;de hücreler O / N inkübe edin. Görselantijen ize: floresan konjüge keçi anti-tavşan IgG ikincil antikor (1 saat, 1/1, 000) ile, inkübasyonu takiben antikor kompleksi. PBS/0.1% Triton X-100 (3 x 5 dk) ile yıkanarak bağlanmamış ikinci antikor çıkarın. </li><li> 4 &#39;,6-diamidino-2-fenilindol / PBS (DAPI; 0.1 ug / mL) ile leke hücreleri. Hücre çekirdeği görselleştirmek için CX3CR1-GFP + / türetilmiş tüm mikroglia - farenin GFP ifade eder. DAPI görselleştirmek için bir 350 nm dalga boyu filtre kullanmak; GFP görselleştirmek için bir 488 nm dalga boyu filtre ve 594 nm dalga boyu filtre p65 ve İba-1 immünokompleksler görselleştirmek için. </li></ol></li><li> Dağı kapak sulu montaj solüsyonu ve floresan mikroskop kullanarak mikroglianın görselleştirmek kayıyor. </li></ol>

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Neuroimmun. Pharmacol. 8, 94-117 (2013).</li><li>Su, X., 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=Synuclein+activates+microglia+in+a+model+of+Parkinson's+disease.">Synuclein activates microglia in a model of Parkinson's disease.</a> Neurobiol. Aging. 29, 1690-1701 (2008).</li><li>Minghetti, L., Levi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+as+effector+cells+in+brain+damage+and+repair:+focus+on+prostanoids+and+nitric+oxide.">Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide.</a> Prog. Neurobiol. 54, 99-125 (1998).</li><li>Hirsch, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+and+Parkinson's+disease.">Glial cells and Parkinson's disease.</a> J. Neurol.. 247 (2), 58-62 (2000).</li><li>Liu, B., 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=Role+of+nitric+oxide+in+inflammation-mediated+neurodegeneration.">Role of nitric oxide in inflammation-mediated neurodegeneration.</a> Ann. NY Acad. Sci. 962, 318-331 (2002).</li><li>Shie, F. S., Nivison, M., Hsu, P. C., Montine, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+microglialinnate+immunity+in+Alzheimer's+disease+by+activation+of+peroxisome+proliferator-activated+receptor+gamma.">Modulation of microglialinnate immunity in Alzheimer's disease by activation of peroxisome proliferator-activated receptor gamma.</a> Curr. Med. Chem. 16, 643-651 (2009).</li><li>Rogers, J., Mastroeni, D., Leonard, B., Joyce, J., Grover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation+in+Alzheimer's+disease+and+Parkinson's+disease:+are+microglia+pathogenic+in+either+disorder.">Neuroinflammation in Alzheimer's disease and Parkinson's disease: are microglia pathogenic in either disorder.</a> Int. Rev. Neurobiol. 82, 235-246 (2007).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <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+murinemicroglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murinemicroglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigenpresentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigenpresentation to myelin basic protein-reactive CD4+ T cells compared.</a> J. Immunol. 154, 4309-4321 (1995).</li><li>Ford, A. L., Foulcher, E., Goodsall, A. L., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+digestion+with+dispase+substantially+reduces+lymphocyte+and+macrophage+cell-surface+antigen+expression.">Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.</a> J. Immunol. Methods. 194, 71-75 (1996).</li><li>Pino, P. A., Cardona, A. E. <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+brain+and+spinal+cord+mononuclear+cells+using+percoll+gradients.">Isolation of brain and spinal cord mononuclear cells using percoll gradients.</a> J. Vis. Exp. , (2011).</li><li>Veremeyko, T., Starossom, S. C., Weiner, H. L., Ponomarev, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microRNAs+in+microglia+by+real-time+PCR+in+normal+CNS+and+during+neuroinflammation.">Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation.</a> J. Vis. Exp. , (2012).</li><li>Su, X., Federoff, H. J., Maguire-Zeiss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-synuclein+overexpression+mediates+early+proinflammatory+activity.">Mutant alpha-synuclein overexpression mediates early proinflammatory activity.</a> Neurotox. Res. 16, 238-254 (2009).</li><li>Jung, 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=Analysis+of+fractalkine+receptorCX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptorCX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Imai, T., 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=Identification+and+molecular+characterization+of+fractalkine+receptor+CX3CR1,+which+mediates+both+leukocyte+migration+and+adhesion.">Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.</a> Cell. 91, 521-530 (1997).</li><li>Cardona, A. 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=Control+of+microglial+neurotoxicity+by+the+fractalkine+receptor.">Control of microglial neurotoxicity by the fractalkine receptor.</a> Nat. Neurosci. 9, 917-924 (2006).</li><li>Streit, W. J., Walter, S. A., Pennell, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+microgliosis.">Reactive microgliosis.</a> Prog. Neurobiol. 57, 563-581 (1999).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus:immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li></ol>

Yazarlar araştırma ilgi potansiyel bir çatışma şeklinde olabilir herhangi bir ticari veya finansal ilişkilerin yokluğunda yapılmış olduğunu beyan ederim.

Bu prosedür, neonatal farelerde kortikal mikroglia izolasyonu için etkili bir yöntem sunmaktadır. Bu prosedür, flüoresan görüntüleme, bağışıklık kimyası ve ELISA ile tespit edildiği üzere, 1) koruyucu mikroglia immunfenotipik ve işlevsellik iki kat yararı vardır, ve 2) mikroglia önce diğer glial hücreleri (astrositler ve oligodentrocytes) varlığında, olgun izin glial kültür olgunlaşma döneminde önemli hücre-hücre etkileşimleri kolaylaştırabilir izolasyon. Önemlisi, izole hücreler ko...

Mikroglial hücreleri, CNS parankima gözetim makrofajlar, erişkin memeli beyin, toplam hücre popülasyonunun yaklaşık olarak% 12 ihtiva eder. Mikroglia yolk kesesi miyeloid öncü hücrelerinden türetilen ve yetişkin CNS 1-5 içinde farklı bölgelerde cytoarchitectural hücre yoğunluğu ve morfolojik olarak değişmektedir. Sağlıklı bir yetişkin beyninde, mikrogliya ince, dinamik süreçleri ile, küçük dallanmış veya polar hücrelerdir. Bununla birlikte, in vivo görüntüleme çalışmaları mikroglial işlemleri dinamik &quot;andıran bir şekilde uzanır ve mikro izlemek için geri göstermektedir; çevresel makrofaj morfolojisi aksine, mikroglia hücresel işlem olarak görünebilir sağlıklı beyinlerinde bir hareketsiz, düşük profilli bir fenotip göstermektedir örnekleme ve &quot;6,7 ölçme. Mikroglia geçiş, yüksek ve diferansiyel olarak beyinde çevresel ve patofizyolojik değişikliklere duyarlısırasıyla bir efektör devlet-genellikle kendi dinlenme olarak kabul ve aktif devletler, bir surveillant dan. Aktivasyonuna Bu anahtar, bu patojenle ilişkili moleküler kalıpları yanıt toll benzeri reseptörler (TLR), (PAMPs), yani bakteri ve viral türevli lipoproteinler olarak membrana bağlı görüntü tanıma reseptörleri (PRR) arasında takılma ile aracılık edilebilir , nükleik asitler, karbonhidratlar ve 8-11. PAMPs ek olarak, PRR&#39;ler, aynı zamanda, hücre hasarı 12-16 gibi CNS homeostatisinde sarsımına temsil tehlike / hasar ile bağlantılı moleküler kalıpları (DAMPS) olarak bilinen steril, patojenik olmayan moleküllere karşı, mikroglial aktivasyonunu teşvik ettiği gösterilmiştir. Bir kez yerleştikten sonra PRR mikroglial hücre morfolojisi ve gen ekspresyonunda değişiklikler ile sonuçlanır bir hücre içi sinyalleme kaskadını başlatabilir, özel olarak ise, aktive edilmiş mikroglia bir amoeboid benzeri fenotip adapte hücre çekirdeğine NF-KB p65 alt ünitesi (p65) yerini değiştirmek ve upregüle süredenşeklinde gösterilmektedir ve bu reaktif oksijen türleri (ROS) 16-24 ile birlikte, tümör nekroz faktörü-α (TNF-α), interlökin-1 β (IL-1β) gibi proenflamatuar sitokinlerin salgılanması. CNS&#39;nin doğuştan gelen immün yanıt olarak tamamlayıcı olsa da, bu salgılanan moleküller de bu şekilde, Parkinson ve Alzheimer hastalığı gibi hastalık durumlarının 25-29 nörodejenerasyonu neden olan ve arttıran, nöronal oksidatif stres geliştirmek için bulunmuştur. Bununla birlikte, patolojik durumların mikrogliyal aktivasyonun mekanizması tam olarak anlaşılmış değildir. Mikroglial aktivasyonun vivo özellikleri birçok kültür değinmeyecek olabilir Bu nedenle, mikroglia izolasyonu, bu biyolojik süreçlere güçlü bir araştırma aracıdır. Çeşitli yöntemler CNS doku 30,31 enzimatik sindirimi takip eden bir Percoll gradyanı ile izolasyon dahil olmak üzere, mikroglia, izole edilmesi için kullanılabilir. Bununla birlikte, enzimatik sindirim değiştirebilirAntijen ekspresyonu 32 hücre yüzeyi azaltılması ve burada tarif edilen yönteme göre, hayvan başına daha düşük hücre verimle sonuçlar ile hücrelerin immünofenotipi. Daha önce bir Percoll verim 3-5 x 10 5 hücre 30,33,34 yoluyla tüm CNS izolasyon yöntemleri rapor ederken Özellikle, biz, 7,5 x 10 5 hücre yavru korteksinde başına ortalama mikrogliya verim rapor. Bu prosedür, bu şekilde mikroglial immünfenotip ve işlevselliğini korumak, düşük-yapışma özelliklerine göre mikroglia izole edilmesi ile sindirim enzimlerinin aşılmaktadır. Döner bir karıştırıcı, daha önce bir uzantısına mekanik ajitasyon ile, ve C57BL / 6 faregiller korteks - Bu çalışmada, neonatal heterozigot CX3CR1-GFP (CX3CR1-GFP + /) türetilmiş karışık glial kültürlerinden mikroglial hücreleri izole edilmesini tarif 24,35 yayınlanan yöntem. Biz mikrogliya kolay görsellik için eski fare zorlanma kullanmak gibibir monosit spesifik promoter 36-38 - Bu farenin endojen CX3CR1 mahalinin kontrolü altında GFP ifade eder. Bu yöntem, bir korunmuş immünofenotipi ex vivo bakteriyel lipopolisakkarid (LPS) veya Pam 3 CSK 4 ile meydan morfolojik değişiklikler, p65 nükleer translokasyonu ve TNF-α salgılanması ile gösterildiği gibi, TLR4 ve TLR1 / 2 agonistleri ile son derece saf bir kültür Mikroglial üretir sırasıyla.

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="160">
			<td height="160" style="height:160px;width:233px;">Glucose</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:143px;">Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &#176;C; shelf life: 3-6 months. Used to make MCM and MGM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Sodium pyruvate 100 mM (100x)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30239.01</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Penicillin/Streptomycin 10,000 units/ml (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15140-122</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM, MGM, MM, and DM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">L-glutamine 200 mM (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">25030-081</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Fetal Bovine Serum (Defined)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30070.03</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:233px;">Minimum Essential Medium Earle&#39;s (MEM)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">15-010-CV</td>
			<td style="width:143px;">Without L-glutamine. Contains Earle&#39;s salts. Used to make MCM, MGM, and DM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Horse Serum</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">16050</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">21-021-CV</td>
			<td style="width:143px;">Without calcium and magnesium. Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">HEPES 1 M</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15630-031</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T-75 Flask</td>
			<td style="width:149px;">Corning</td>
			<td style="width:155px;">430641</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">4&#39;,6-Diamidino-2-Phenylindole, dilactate (DAPI)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">D3571</td>
			<td style="width:143px;">Used to stain cell nucleus.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-Iba1</td>
			<td style="width:149px;">Wako</td>
			<td style="width:155px;">019-19741</td>
			<td style="width:143px;">Used at 1/750 dilution for ICC staining of Iba1.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-NF&#954;B (p65)</td>
			<td style="width:149px;">Abcam</td>
			<td style="width:155px;">7970</td>
			<td style="width:143px;">Used at 1/1250 dilution for ICC staining for p65.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">Alexafluor 594 Goat anti-Rabbit IgG (H+L)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">A11012</td>
			<td style="width:143px;">Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">10 ml Disposable Serological Pipet</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">13-678-11E</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">50 ml Disposable Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-8</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">15 ml Disposible Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-12</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Sterile Polystyrene Petri Dish</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">875713</td>
			<td style="width:143px;">100 mm x 15 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Straight Metzembaum (scissor #1)</td>
			<td style="width:149px;">Roboz Surgical</td>
			<td style="width:155px;">RS-6010</td>
			<td style="width:143px;">1; 5 inch; used for removing head</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Scissor: Vannas 
			(scissor #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">15000-08</td>
			<td style="width:143px;">1; non-angled; 2.5mm cutting edge; used to open scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Student Vannas (scissor #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91501-09</td>
			<td style="width:143px;">1; curved; used to mince brain tissue</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Forcep: Dumont #7 
			(forcep #1)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91197-00</td>
			<td style="width:143px;">2; used to secure nose and remove cortices&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #2 
			(forcep #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11223-20</td>
			<td style="width:143px;">1; used to remove scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #3 
			(forcep #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11231-30</td>
			<td style="width:143px;">1; used to remove skull</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #5a 
			(forcep #4)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11253-21</td>
			<td style="width:143px;">1; used to remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table of specific equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Name of Equipment</td>
			<td style="width:149px;">Name of Company</td>
			<td style="width:155px;">Catalogue Number</td>
			<td style="width:143px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Zoom Stereo Dissection Microscope&#160;</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:155px;">SZ4060</td>
			<td style="width:143px;">Microscope is placed inside Laminar-Horizontal Flow Cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Laminar-Horizontal Flow Cabinet</td>
			<td style="width:149px;">Nuaire</td>
			<td style="width:155px;">NU-201-330</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Biological Safety Cabinet</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:155px;">3440001</td>
			<td style="width:143px;">Class II</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:233px;">Water-Jacketed CO2 Incubator</td>
			<td style="width:149px;">VWR</td>
			<td>97025-836</td>
			<td style="width:143px;">Set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out buckets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006441</td>
			<td style="width:143px;">To be used with Swing-out rotor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out Rotor</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006445</td>
			<td style="width:143px;">Max Radius: 19.2 (cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Sorvall Legend RT+ Centrifuge 
			(clinical centrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75-004-377</td>
			<td style="width:143px;">With swing-out rotor</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">AccuSpin Micro 17 microcentrifuge 
			(tabletop microcentrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>S98645</td>
			<td style="width:143px;">With microliter rotor (24 x 1.5/2.0 ml; 
			Cat #: 75003524)&#160;&#160;</td>
		</tr>
	</tbody>
</table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

CNS dokusundan mikrogliya İzolasyonu ex vivo mikrogliyal biyolojisi okumak için kullanılan güçlü bir araştırma aracıdır. Mevcut yöntem, döner bir karıştırıcı, mekanik çalkalama ile neonatal murin kortekslerinden mikroglia izolasyonu için bir prosedürü göstermektedir. Bu yöntem, mikroglia izolasyon verimi canlı içinde normal ve patolojik olmayan koşullarda sakin mikroglia göstergesidir morfolojik ve fonksiyonel özelliklerini gösteren yüksek ölçüde saf bir kortikal mikroglia. Morfolojik değişiklikler, NF-KB (p65) bir p65 alt birimi nükleer translokasyonu ve ayırt edici özelliği pro-enflamatuar sitokin salgılanması, tümör nekroz faktörü-α (TNF-α endüksiyonu ile gösterildiği gibi, bu prosedür, aynı zamanda mikroglial immünfenotip ve biyokimyasal özelliğe korur ), lipopolisakarid&#39;e (LPS) ve Pam 3 CSK 4 (Pam) zorluklar üzerine. Bu yüzden, bu izolasyon prosedürü ve hareketsiz acti hem de immünfenotip korurex vivo koşullarda mikrogliya biyolojiyi araştıran deneysel bir yöntem sağlayan çıkartılır mikrogliya.

Izolasyonu sırasında mikroglia ex vivo immunfenotipine ve işlevselliğini İstinat mikrogliyal biyoloji için bir soruşturma model olarak bu hücreleri kullanmak edebilmek için önemlidir. (- Ve C57BL / 6 CX3CR1-GFP + /) ve LPS veya Pam ya ile tedavi edilen kültürler, bu yöntem kullanılarak mikroglia immunofunctionality başarılı bir şekilde korunması göstermek amacıyla, P3 yenidoğanlardan kortikal mikroglia izole edilmiştir. Şekil 1A&...

Watch this Scientific Journal Video about Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates at JoVE.com

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Mikrogliyal biyoloji başarılı bir soruşturma için bir anahtar CNS dokudan izolasyonu sırasında ex vivo mikroglialardan immunofunction korunmasıdır. Flüoresan görüntüleme, bağışıklık kimyası ve proenflamatuar stimuli lipopolisakarit (LPS) ile Pam 3 CSK 4 (PAM) ile ELISA aşağıda mikroglia aktivasyonu ile değerlendirilen gibi son derece saf ve immunofunctional hücre kültürlerinde döner çalkalama sonuçları ile mikroglia izole edilmesi.

Kemirgen Yenidoğan itibaren Korunan immunfenotipine ve İşlevselliği ile Kortikal mikrogliya İzolasyonu

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

isolation-cortical-microglia-with-preserved-immunophenotype

Research

JoVE Journal

Immunology and Infection

15.7K Görüntüleme.  Georgetown University Medical Center.  Mikrogliyal biyoloji başarılı bir soruşturma için bir anahtar CNS dokudan izolasyonu sırasında ex vivo mikroglialardan immunofunction korunmasıdır. Flüoresan görüntüleme, bağışıklık kimyası ve proenflamatuar stimuli lipopolisakarit (LPS) ile Pam 3 CSK 4 (PAM) ile ELISA aşağıda mikroglia aktivasyonu ile değerlendirilen gibi son derece saf ve immunofunctional hücre kültürlerinde döner çalkalama sonuçları ile mikr...

Video: Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Yenidoğan Rat Beyin Dokusu Karışık Glial Hücre Kültürleri Primer mikroglia İzolasyonu

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

İmmünoloji ve Enfeksiyon

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 ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Yenidoğan ve Erişkin Merkezi sinir sisteminin Kültüre Mikroglia

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

İzolasyon ve Farklı Yaşlı Fare Timik Greft Nakli.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Maternal Fetal Arayüz at Fare Dokular lökosit İzolasyonu

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

İzolasyon ve murin lenfositlerin aktivasyonu

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Bir kullanma Lenfosit Ekstravazasyonunun analizi<em&gt; İn Vitro</em&gt; İnsan Kan-beyin bariyeri Model

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

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Kemirgen Yenidoğan itibaren Korunan immunfenotipine ve İşlevselliği ile Kortikal mikrogliya İzolasyonu

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