Bu çalışma, Agence Nationale pour la Recherche (ANR-12-MALZ-0003-02-P2X7RAD), Association France Alzheimer ve Bpifrance&#39;in hibeleriyle desteklendi. Laboratuarımızda ayrıca Inserm, CNRS, Université Pierre et Marie-Curie ve &quot;Investissements d&#39;avenir&quot; ANR-10-IAIHU-06 (IHU-A-ICM) programı desteklenmektedir. CELIS hücre kültürü çekirdeğinin desteğine teşekkür ediyoruz.

Burada açıklanan tüm yöntemler, ICM Enstitüsünde Kurumsal Hayvan Bakımı ve Kullanım Komitesi tarafından ve Darwin Fransız Etik Hayvan Komitesi tarafından onaylanmıştır ve 01407.02 protokolüyle kapsanmıştır. 1. Hazırlık <ol><li> Sindirim kokteylini her fare için aşağıdakileri birleştirerek 1.5 mL&#39;lik bir tüp içerisinde hazırlayın: 1 mL Fosfat Tamponlu Salin (PBS); 13 wunsch / mL&#39;de (stok çözeltisi) 123 uL sindirim enzimi (materyal tablosuna bakınız), son konsantrasyon: 1.6 w / sn; Ve 100 mg / mL&#39;de (stok çözeltisi) 5 uL DNaz I (bakınız tablo), nihai konsantrasyon 0.5 mg / mL. </li></ol> 2. Perfüzyon ve Diseksiyon <ol><li> Fareleri ölümcül bir pentobarbital dozu (400 mg / mL&#39;de 100 μL) ile euthanize edin. </li><li> Hayvanı yatırın ve vücudu% 70 etanol ile ıslatın. </li><li> Göğüs boşluğunu açığa çıkarmak için ventral cildi, xyphoid prosesin hemen altındaki ince makaslarla kesin.y. </li><li> Diyaframa bir kesi yapın ve kalbi ortaya çıkarmak için (ve diğer organları kesmekten kaçınmak için) rostral yönde kaudal bir şekilde ince makasla göğüs kafesinin yanal yönlerini kesin. Sol ventrikül (LV) ve sağ atriyumun (RA) tanımlanması. </li><li> LV&#39;nin içine 25 G iğne ile bir kelebek kateteri takın. İnce makaslarla RA&#39;ya bir kesi yapın ve kan akışının başlangıcında, damarlardan hücreleri ve kanları çıkarmak için 20 mL PBS ile LV boyunca 10 mL / dakika akış oranında perfüzyona başlayın; Başarılı bir perfüzyon akciğerlerin ve karaciğerin kanlanması nedeniyle göğüs gerilmesi ile belirtilir. </li><li> Hayvanı orta makasla dekapatlayın. Omurga sütununu ince makaslarla parçalara ayırın ve omuriliğin ventral tarafındaki karın duvar kaslarını çıkararak ve kuyruk tabanındaki vertebral kolonu keserek vücudundan ayırın. </li><li> PBS içeren bir 10 mL&#39;lik şırınga yerleştirin ve bel desteğine 200 ml&#39;lik bir pipet yerleştirin.Omurga kolu ve omurili servikal taraftan boşaltın. NOT: 200 mcL pipet ucu en geniş ucunda (yaklaşık 1 cm) makasla kesilir ve daha önce PBS ile dolu 10 mL şırıngaya sıkıca yerleştirilir. </li><li> Kafatasının üstündeki cildi çıkarın ve beyni ortaya çıkarmak için forseps kullanın. Kafatasının tabanına bir makas bıçağı takın ve sagital sütürü takiben kafatayı kesin. Kesime küçük forseps yerleştirin ve kafatasını hafifçe kaldırın. Fare kafasından beyni ayırın ve koku ampulünü ve serebellumu çıkarın. </li><li> Sindirim kokteyli (1. adımdan itibaren) 1.128 mL PBS içeren 1.5 mL&#39;lik bir tüp içerisinde beyin ve omurilik toplayın. </li></ol> 3. Hücre Ayrışımı <ol><li> Küçük makasla 1-2 mm 3 parçaya ( Şekil 1. ) 1,5 mL tüpün (adım 2.9) beyin ve omurili ince şekilde kesin. </li><li> Öğütülmüş dokuları içeren 1.5 mL tübü bir incuba&#39;da 30 dakika inkübe edin37 ° C&#39;de% 5 CO 2 ile yıkayın. NOT: Bu adımda yoğunluk derecesini hazırlayın. </li><li> Enzimatik reaksiyonu durdurmak için 20 ml 0.5 M EDTA (stok solüsyonu), nihai konsantrasyon 10 mM, 1.5 mL tüp ekleyin. </li><li> 5 mL pipetle aşağı yukarı pipetle hafifçe hücre süspansiyonu yapın. </li><li> 10 mL şırınganın pistonu kullanarak 50 mL&#39;lik bir tüpteki hücre süspansiyonunu bir naylon ağ (70 um gözenek boyutu) üzerinden geçirin. </li><li> Naylon örgüyü 20 mL% 5 Fetal Sığır Serumu (FBS) -PBS ile yıkayın. </li><li> Oda sıcaklığında (18 ° C) 7 dakika boyunca (300 xg) santrifüjleyin. Süpernatantı aspirasyon ile atın ve sonraki adıma geçin. Süpernatantı çıkartırken son derece dikkatli olun; Pelet rahatsız etmeyin, pellet, aspirasyon pipetiyle kolaylıkla aspire edilebilir. </li></ol> 4. Yoğunluk Dereceli <ol><li> Yoğunluk gradyan ortamına uygun miktarda 10x PBS ekleyerek yoğunluk gradyanı ortam stok solüsyonu hazırlayın. NOT: Dokuz parçalı yoğunluk gradyanı ortamına bir parça 10X PBS ekleyin. </li><li> Stok yoğunluğu gradyan ortamını Tablo 1&#39;de açıklandığı gibi farklı yüzdeler için 1x PBS ile seyreltin. </li><li> 4 mL% 30 yoğunluk gradyan ortamı ile pelletini (adım 3.7&#39;den) tekrar süspansiyon haline getirin ve 15 mL&#39;lik bir tüpe aktarın. </li><li> 15 mL&#39;lik tüpün altına bir Pasteur pipeti yerleştirin ve yavaşça% 37&#39;lik yoğunluk derecesi ortamının 4 mL&#39;sini altına yerleştirin. </li><li> Yukarıda tarif edildiği gibi 4 mL% 70 yoğunluk dereceli ortam ekleyin. </li><li> Degrade oda sıcaklığında (18 ° C) 40 dakika boyunca (800 xg) santrifüjleyin. Santrifüjün az veya hiç frenle durduğundan emin olun, böylece ara faz rahatsız edilmez. NOT: Boru katmanlı görünecektir. % 70 -% 37 yoğunluk gradyan arayüzünde tabakalanmış hücreler, mononükleize edilen immün hücrelerdir (mikroglia ve makrofaj alt popülasyonları); Üst düzeyler sırasıyla hücre döküntüsü ve miyelindir ( Şekil 2 ). </li><lI&gt; Bir transfer pipeti kullanarak hafifçe miyelin tabakasını çıkarın. </li><li> Temiz bir 15 mL tüp içine makrofaj alt popülasyon hücreleri içeren 3-4 mL% 70-37 yoğunluk gradiyent ara fazı toplayın. </li><li> PBS ile hücreleri 3 kez yıkayın, toplam hacmi 15 mL. Hücrelerin bulunduğu yoğunluk gradyan ortamının PBS ile en az üç kez seyreltildiğinden ve fren &quot;açık&quot; konumdayken 7 dakika (500 xg, 4 ° C) santrifüje tabi tutulduğundan emin olun. </li><li> Hücre etiketleme veya diğer fonksiyonel testler için hücre pelletini 1 mL PBS ile tekrar süspanse edin. </li></ol> 5. Akış Sitometrisi için Hücre Etiketleme <ol><li> Hücreleri (adım 4.10) bir akış sitometri tüpüne aktarın. </li><li> Adım 4.10&#39;da elde edilen her bir hücre örneğine 1 μL sabitlenebilir ölü hücre lekesi reaktifi ekleyin. Işıktan korunarak buz üzerinde 30 dakika inkübe edin. </li><li> Hücreleri bir kez 2 mL PBS ve 5 dakika boyunca santrifüjle yıkayın (300 xg, 4 ° C). </li><li> Süpernatıtıyı çıkarın ve hücre peletini400 uL PBS% 1 BSA,% 0.1 azid. </li><li> Fc reseptörlerini bloke etmek için 100 uL hücreye 1 μg CD16 / CD32 antikoru ekleyin. Buz üzerinde 10-15 dakika inkübe edin, ışığa karşı korunun. </li><li> İntraselüler boyama için hücre permeabilization için birincil antikor karışımı PBS% 1 BSA,% 0.1 azid veya PBS% 1 BSA,% 0.1 azid,% 0.25 saponin hazırlayın. </li><li> Birincil antikor karışımı (2x) 100 uL ekleyin ve buz üzerinde 30 dakika inkübe edin, ışığa karşı koruyun. NOT: Optimum sonuçların elde edilmesi için deney yapılmadan önce tüm antikorlar titrasyona tabi tutulmalıdır. </li><li> 2 mL PBS ile hücreleri yıkayın ve 5 dakika boyunca santrifüjleyin (300 xg, 4 ° C). Bu adımı üç kez tekrarlayın. </li><li> Birincil antikorlar doğrudan bir floroforla konjuge edilmediyse 100 uL ikincil bir antikor ekleyin ve ışıktan korunarak buz üzerinde 30 dakika inkübe edin. </li><li> 2 mL PBS ile hücreleri yıkayın ve 4 ° C&#39;de 300 xg&#39;de 5 dakika santrifüjleyin. Bu adımı üç kez tekrarlayın. </li><li> Tamir etOda sıcaklığında 15 dakika oda sıcaklığında 100 μL% 1 PFA ile yıkanır. NOT: Dikkat: PFA toksiktir. Duman kabininde kullanın ve kişisel koruyucu ekipman giyin. </li><li> 2 mL PBS ile hücreleri yıkayın ve 4 ° C&#39;de 300 xg&#39;de 5 dakika santrifüjleyin. Bu adımı üç kez tekrarlayın. </li><li> 200 uL PBS ile hücre topağı tekrar süspansiyon ve akış sitometri edinimi ve analizi devam edin. Şekil 3&#39;deki geçiş stratejisini izleyin. </li></ol>

<ol>
	<li>Ginhoux, F., 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=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+differentiation+of+microglia.">Origin and differentiation of microglia.</a> Front Cell Neurosci. 7, 45 (2013).</li><li>Ginhoux, F., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+macrophages:+developmental+pathways+and+tissue+homeostasis.">Monocytes and macrophages: developmental pathways and tissue homeostasis.</a> Nat Rev Immunol. 14 (6), 392-404 (2014).</li><li>Schulz, 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=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>London, A., Cohen, M., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+monocyte-derived+macrophages:+functionally+distinct+populations+that+act+in+concert+in+CNS+plasticity+and+repair.">Microglia and monocyte-derived macrophages: functionally distinct populations that act in concert in CNS plasticity and repair.</a> Front Cell Neurosci. 7, 34 (2013).</li><li>Miron, V. E., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+CNS+remyelination.">Macrophages and CNS remyelination.</a> J Neurochem. 130 (2), 165-171 (2014).</li><li>Mildner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+and+non-redundant+roles+of+microglia+and+myeloid+subsets+in+mouse+models+of+Alzheimer's+disease.">Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer's disease.</a> J Neurosci. 31 (31), 11159-11171 (2011).</li><li>Boillee, 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=Onset+and+progression+in+inherited+ALS+determined+by+motor+neurons+and+microglia.">Onset and progression in inherited ALS determined by motor neurons and microglia.</a> Science. 312 (5778), 1389-1392 (2006).</li><li>Yamasaki, R., 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=Differential+roles+of+microglia+and+monocytes+in+the+inflamed+central+nervous+system.">Differential roles of microglia and monocytes in the inflamed central nervous system.</a> J Exp Med. 211 (8), 1533-1549 (2014).</li><li>Wu, D. 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=Blockade+of+microglial+activation+is+neuroprotective+in+the+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine+mouse+model+of+Parkinson+disease.">Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease.</a> J Neurosci. 22 (5), 1763-1771 (2002).</li><li>Cartier, N., Lewis, C. A., Zhang, R., Rossi, F. 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+role+of+microglia+in+human+disease:+therapeutic+tool+or+target?.">The role of microglia in human disease: therapeutic tool or target?.</a> Acta Neuropathol. 128 (3), 363-380 (2014).</li><li>Ajami, B., Bennett, J. L., Krieger, C., McNagny, K. M., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltrating+monocytes+trigger+EAE+progression,+but+do+not+contribute+to+the+resident+microglia+pool.">Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.</a> Nat Neurosci. 14 (9), 1142-1149 (2011).</li><li>Funk, N., 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+peripheral+hematopoietic+stem+cells+and+monocytes+in+Parkinson's+disease.">Characterization of peripheral hematopoietic stem cells and monocytes in Parkinson's disease.</a> Mov Disord. 28 (3), 392-395 (2013).</li><li>Butovsky, O., 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=Modulating+inflammatory+monocytes+with+a+unique+microRNA+gene+signature+ameliorates+murine+ALS.">Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS.</a> J Clin Invest. 122 (9), 3063-3087 (2012).</li><li>Lewis, C. A., Solomon, J. N., Rossi, F. M., Krieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+cells+in+the+central+nervous+system+of+a+mouse+model+of+amyotrophic+lateral+sclerosis+are+associated+with+blood+vessels+and+express+CX(3)CR1.">Bone marrow-derived cells in the central nervous system of a mouse model of amyotrophic lateral sclerosis are associated with blood vessels and express CX(3)CR1.</a> Glia. 57 (13), 1410-1419 (2009).</li><li>Sedgwick, J. D., 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=Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system.">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proc Natl Acad Sci U S A. 88 (16), 7438-7442 (1991).</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+antigen+presentation+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 antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154 (9), 4309-4321 (1995).</li><li>Martin, E., Boucher, C., Fontaine, B., Delarasse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+inflammatory+phenotypes+of+microglia+and+monocyte-derived+macrophages+in+Alzheimer's+disease+models:+effects+of+aging+and+amyloid+pathology.">Distinct inflammatory phenotypes of microglia and monocyte-derived macrophages in Alzheimer's disease models: effects of aging and amyloid pathology.</a> Aging Cell. , (2016).</li><li>Bennett, M. L., 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=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proc Natl Acad Sci U S A. 113 (12), E1738-E1746 (2016).</li><li>Jin, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+glutamine+transporter+SNAT1+in+Rett+syndrome+microglia:+a+mechanism+for+mitochondrial+dysfunction+and+neurotoxicity.">Dysregulation of glutamine transporter SNAT1 in Rett syndrome microglia: a mechanism for mitochondrial dysfunction and neurotoxicity.</a> J Neurosci. 35 (6), 2516-2529 (2015).</li><li>Bedi, S. S., Smith, P., Hetz, R. A., Xue, H., Cox, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomagnetic+enrichment+and+flow+cytometric+characterization+of+mouse+microglia.">Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.</a> J Neurosci Methods. 219 (1), 176-182 (2013).</li><li>Nikodemova, M., Watters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> J Neuroinflammation. 9, 147 (2012).</li><li>Mizutani, 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=The+fractalkine+receptor+but+not+CCR2+is+present+on+microglia+from+embryonic+development+throughout+adulthood.">The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.</a> J Immunol. 188 (1), 29-36 (2012).</li><li>Lecoeur, H., Ledru, E., Gougeon, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytofluorometric+method+for+the+simultaneous+detection+of+both+intracellular+and+surface+antigens+of+apoptotic+peripheral+lymphocytes.">A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.</a> J Immunol Methods. 217 (1-2), 11-26 (1998).</li><li>Richter, N., 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=Glioma-associated+microglia+and+macrophages/monocytes+display+distinct+electrophysiological+properties+and+do+not+communicate+via+gap+junctions.">Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions.</a> Neurosci Lett. 583, 130-135 (2014).</li><li>Mahad, D., 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=Modulating+CCR2+and+CCL2+at+the+blood-brain+barrier:+relevance+for+multiple+sclerosis+pathogenesis.">Modulating CCR2 and CCL2 at the blood-brain barrier: relevance for multiple sclerosis pathogenesis.</a> Brain. 129 (Pt 1), 212-223 (2006).</li></ol>

Yazarların ifşa edecekleri bir şeyleri yok

Mikroglia ve MDM&#39;nin MSS&#39;de farklı işlevlere ve fenotiplere sahip oldukları gösterildi ve bu nedenle bu makrofaj alt popülasyonlarının tanımlanması ve analizi, nörolojik hastalıkları daha iyi anlamak için gereklidir 9 , 18 , 25 . İki belirteç (CD11b ve CD45) kullanılarak yapılan akış sitometrisi analizi, her alt popülasyon arasındaki ayrımı mümkün kılar ( Şekil 3C...

Mikrogram, merkezi sinir sisteminin (MSS) parenkimal dokuya ait makrofajlarıdır. İki önemli fonksiyonel rol oynuyorlar: Bağışıklık savunması ve SSS homeostazisinin korunması. Kemik iliğindeki hematopoietik kök hücrelerden sürekli olarak yenilenen MDM&#39;den farklı olarak, mikroglial hücreler embriyonik gelişme sırasında beyinde koloni oluşturan yolk kesesinden (YS) kaynaklanan ilkel hematopoietik progenitör hücrelerden ayırırlar 1 , 2 , 3 . Kemirgenlerde, transkripsiyon faktörü Myb, tüm kemik iliği kaynaklı monositlerin ve makrofajların gelişiminde çok önemli bir rol oynamaktadır, ancak YS türevi mikroglia için bu faktör gerekli değildir ve farklılaşma, transkripsiyon faktörü PU.14&#39;e bağımlıdır. Sağlıklı CNS&#39;de, mikroglia, çevrede sürekli olarak örnek olan dinamik hücrelerdir, scaPatojenleri veya doku hasarını işlemek için nning ve survey 5 . Bu tür sinyaller algılanması, yaralanmanın çözüm yolunu başlatır. Mikroglia, hızlıca ayrılmış bir morfolojiden amoeboid bir fogositoza ve ardından pro-veya anti-inflamatuar sitokinler gibi çeşitli mediatörlerin salınmasını izler. Böylece, mikro çevreye bağlı olarak aktive edilmiş mikroglia farklı priming durumlarından 6 oluşan bir spektrum kazanabilir. Mikroglia, birçok nörolojik rahatsızlığın gelişimini ve ilerlemesini derinden etkilemektedir. Alzheimer hastalığının (AD) 7 , Amyotrofik Lateral Skleroz (ALS) 8 , Multipl Skleroz (MS) 9 veya Parkinson hastalığının (PD) 10 kemirgen modellerinde mikroglia&#39;nın zararlı nörotoksisite indükleyen ya da etki eden iki rol oynadığı gösterilmiştir Nöroprotektif bir tarzda, bağımlı olanSpesifik hastalık, hastalık evresi ve hastalığın sistemik bağışıklık bölmesinden 7 , 8 , 9 , 10 , 11 etkilenip etkilenmediği. Yukarıda belirtilen hastalıklarda gözlenen CNS lezyonlarının çoğunda sadece parankimal mikrogram değil aynı zamanda perivasküler ve meningeal makrofajların yanı sıra CNS-sızıntılı MDM de dahil olmak üzere heterojen bir myeloid hücre popülasyonu bulunur. Bu hücre tipleri, yaralanma ve onarım ile ilişkili patofizyolojik mekanizmalara farklı şekilde katkıda bulunabilir 7,12,13,14,15. Bu hastalık modellerini inceleyen araştırmacılar için mevcut zorluk, periferik monositlerin ve makrofajların MSS&#39;ye sızdığını ve eğer öyleyse distBu hücrelerden ikamet eden mikrogliaları kucaklayın. Aslında mikroglial hücreler çok plastiktir; Aktive edildiğinde, mikrogram genellikle periferik monositler ve makrofajlar tarafından eksprese edilen belirteçleri yeniden ifade eder. Bu nedenle, konu, yerleşik mikroglianın sızma monositleri ve makrofajları ayırt edebilen belirteçlerin belirlenmesine dayanır. Bu popülasyonların beyin dilimleri üzerindeki immünohistolojik uygulamaları ile ayrımcılık spesifik antikorların olmaması nedeniyle sınırlıdır. Bununla birlikte, akış sitometrisi analizi, çeşitli belirteçlerin ekspresyonunu değerlendirmek ve hücre popülasyonlarını (örneğin, lenfositler, makrofajlar / MDM CD11b + CD45 yüksekleri ve mikroglia CD11b + CD45 med ) ayırmak için etkili bir teknik yanı sıra hücre alt popülasyonları 16 , 17 , 18 . Bu protokol,Optimize edilmiş, enzimatik bir doku ayrışması ve bir yoğunluk gradiyentli santrifüjleme kullanarak nörolojik hastalık modellerinde fare CNS&#39;den alınan mononükleer hücreler; Hem de flow sitometri kullanarak CNS&#39;de mikroglia ve MDM popülasyonlarını ayırt etmek için bir yöntem. Bir diğer yaklaşım, miyelin&#39;i yok etmek ve spesifik antikorlara 19 , 20 , 21 konjuge edilmiş manyetik boncuklar kullanarak hücreleri saflaştırmaktır. Anti-miyelin manyetik boncuklar kullanan miyelin giderimi daha pahalıdır ve canlılığı ve izole edilmiş hücrelerin verimi 22&#39;yi etkiler. Bu adım ve mikroglianın izleyen immünomanyetik ayrımı, spesifik bağışıklık hücre popülasyonları 21 , 22 için ileri araştırmaları sınırlandırır. Bu prosedürler, hastalık gelişiminde makrofaj alt popülasyonlarının incelenmesi için kolay bir yol sağlar veMakrofaj fenotipleri ve aktivasyon durumlarındaki ilaç etkilerini veya gen modifikasyonlarını belirlemek.

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5-month-old Mice</td>
			<td style="width:223px;">Janvier</td>
			<td style="width:119px;">C57BL/6J</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Liberase TL Research Grade</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">5401020001</td>
			<td>Digestion enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Deoxyribonuclease I from bovine pancreas</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Percoll</td>
			<td style="width:223px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0891-01&#160;</td>
			<td>Density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell Strainer size 70 &#181;m Nylon</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">731751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Venofix 25G</td>
			<td style="width:223px;">BRAUN</td>
			<td style="width:119px;">4056370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Piston syringe 10 mL</td>
			<td style="width:223px;">Terumo</td>
			<td style="width:119px;">SS+10ES1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Pasteur pipette 230 mm</td>
			<td style="width:223px;">Dustcher</td>
			<td style="width:119px;">20420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">1,5 mL&#160; tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">0030 123.328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">15 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">50 mL&#160; tube</td>
			<td style="width:223px;">TPP</td>
			<td style="width:119px;">91050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">5 mL polystyrene round bottom tube</td>
			<td style="width:223px;">BD Falcon</td>
			<td style="width:119px;">352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (1X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">D-PBS (10X) without Ca2+/Mg2+</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14200-067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fetal bovine serum</td>
			<td style="width:223px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Bovine Serum Albumin solution 30%</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Paraformaldehyde 32% Solution</td>
			<td style="width:223px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714-S</td>
			<td>Caution -Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Saponin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Sodium Azide</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">45-4031&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat anti-mouse CD45 (clone 30-F11)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">563890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">BV421 Rat IgG2b, &#954; Isotype Control RUO</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">562603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rabbit anti-mouse TMEM119 (clone28-3)</td>
			<td style="width:223px;">Abcam</td>
			<td style="width:119px;">ab209064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">AlexaFluor 647 Donkey anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Anti-Mouse CD16/CD32 Purified</td>
			<td style="width:223px;">eBioscience</td>
			<td style="width:119px;">14-0161</td>
			<td>Mouse Fc Block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fixable Dead Cell Stain Kits</td>
			<td style="width:223px;">Invitrogen</td>
			<td style="width:119px;">L34969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CCR2 APC-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5538A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rat IgG2B APC-conjugated Isotype Control</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC013A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse CX3CR1 PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">FAB5825P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Goat IgG PE-conjugated Antibody</td>
			<td style="width:223px;">R&#38;D</td>
			<td style="width:119px;">IC108P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Centrifuge</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:119px;">5804R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Cell analyzer</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:119px;">BD FACSVERSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Data Analysis Software</td>
			<td style="width:223px;">FlowJo LLC</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fine scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14090-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Standard Pattern Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11000-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mayo Scissors</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">14010-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Dumont #5 Forceps</td>
			<td style="width:223px;">F.S.T</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry

Birçok çalışma, merkezi sinir sistemi (CNS) patolojilerinde bağışıklık hücrelerinin, özellikle makrofajların rolünü göstermiştir. CNS&#39;de iki ana makrofaj popülasyonu vardır: (i) MSS&#39;nin yerleşik makrofajları olan ve embriyogenez sırasında sarı kese öncülerinden türetilen mikroglia ve (ii) sızıntı yapabilen monosit türevi makrofajlar (MDM) Hastalık sırasında MSS&#39;dir ve kemik iliği progenitörlerinden türemiştir. Her makrofaj alt popülasyonunun rolü, incelenen patolojiye göre değişir. Dahası, histolojik belirteçler veya bu makrofaj alt popülasyonları için kullanılan ayırt edici kriterler üzerinde fikir birliği bulunmamaktadır. Bununla birlikte, akış sitometrisi ile CD11b ve CD45 markörlerinin ekspresyon profillerinin analizi, mikrogliayı (CD11b + CD45 med ) MDM&#39;den (CD11b + CD45 yüksek ) ayırmamızı sağlar. Bu protokolde, yoğunluk gradyanı santrifüjününVe akış sitometri analizi bu CNS makrofaj alt popülasyonlarını karakterize etmek için ve yakınlarda yayınladığımız gibi bu hücreler tarafından ifade edilen birkaç ilgi işaretçisini incelemek için kullanılabilir. Bu nedenle bu teknik, nörolojik hastalıkların fare modellerinde makrofajların rolünü daha iyi anlayabilir ve ayrıca bu hücreler üzerindeki ilaç etkilerini değerlendirmek için de kullanılabilir.

Yoğunluk gradyanlı santrifüjleme ve antikor boyama işleminden sonra hücreler bir akış sitometresinde tutuldu ve aşağıdaki gibi bir morfolojik geçiş stratejisi kullanılarak analiz edildi. Tek hücrelerin çiftleşmelerden ayırt edilmesi için, İleri-Dağılmış-Alan (FSC-A) ve İleri-Dağılmış Yükseklik (FSC-H) karşılaştırması için bir ilk kapı tanımlanmıştır ( Şekil 3A ). Tek hücreler daha sonra göreceli hücre büy...

Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Bu protokol, akış sitometrisi ile yetişkin fare merkezi sinir sisteminde makrofaj alt popülasyonlarının bir analizini sağlar ve bu hücrelerin eksprese ettiği çoklu belirteçlerin çalışması için yararlıdır.

Akış Sitometresi ile Merkezi Sinir Sisteminden Mikroglia ve Monosit Türevi Makrofajların Analizi

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

analysis-microglia-monocyte-derived-macrophages-from-central-nervous

Research

JoVE Journal

Immunology and Infection

23.6K Görüntüleme.  Inserm U 1227, CNRS UMR 7225.  Bu protokol, akış sitometrisi ile yetişkin fare merkezi sinir sisteminde makrofaj alt popülasyonlarının bir analizini sağlar ve bu hücrelerin eksprese ettiği çoklu belirteçlerin çalışması için yararlıdır. 

Video: Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

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 ...

İmmünoloji ve Enfeksiyon

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Makrofaj koloni stimüle edici faktör farklılaştırılmış insan monosit türevli makrofajlar Kültür

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Görüntüleme Sitometrisi ile İnsan Monosit türetilmiş dendritik hücrelerle Karakterizasyonu: İki Monosit İzolasyon Protokoller Arasında Bir Karşılaştırma

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Akış Sitometrisi ile Sıçan Böbrek gelen Makrofagların Fenotipik Karakterizasyonu

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Yalıtım, karakterizasyonu ve obezite ile ilgili iltihap tarafından etkilenen dokular makrofajlar arıtma

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived 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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

İnsan monosit alt kümeleri tam kan tarafından karakterizasyonu Akış Sitometresi Analizi

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Deneysel Otoimmün Ensefalomiyelit Sırasında Merkezi Sinir Sisteminde Lenfosit Infiltrasyonunun Akış Sitometrik Analizi

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

İmmün hücrelerin Aşağı Akış Analizi için Merkezi Sinir Sistemi Dokularının ve İlişkili Meninkslerin İzole Edilmesi

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Akış Sitometri Analizi için Fare Böbrek-Resident CD8+ T hücrelerinin izolasyonu

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...