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><tbody><tr><td>5 month-old Mice</td><td>Janvier</td><td>C57BL/6J</td><td></td></tr><tr><td>Liberase TL Research Grade</td><td>Sigma-Aldrich</td><td>5401020001</td><td>Digestion enzyme</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare Life Sciences</td><td>17-0891-01</td><td>Density gradient medium</td></tr><tr><td>Cell Strainer size 70 &amp;micro;m Nylon</td><td>Corning</td><td>731751</td><td></td></tr><tr><td>Venofix 25 G</td><td>BRAUN</td><td>4056370</td><td></td></tr><tr><td>Piston syringe 10 mL</td><td>Terumo</td><td>SS+10ES1</td><td></td></tr><tr><td>Pasteur pipette 230 mm</td><td>Dustcher</td><td>20420</td><td></td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>0030 123.328</td><td></td></tr><tr><td>15 mL tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>50 mL tube</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube</td><td>BD Falcon</td><td>352054</td><td></td></tr><tr><td>D-PBS (1x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>D-PBS (10x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14200-067</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>Bovine Serum Albumin solution 30%</td><td>Sigma-Aldrich</td><td>A7284</td><td></td></tr><tr><td>Paraformaldehyde 32% Solution</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Caution -Toxic</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td><td>eBioscience</td><td>45-0112</td><td></td></tr><tr><td>Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td><td>eBioscience</td><td>45-4031</td><td></td></tr><tr><td>BV421 Rat anti-mouse CD45 (clone 30-F11)</td><td>BD Biosciences</td><td>563890</td><td></td></tr><tr><td>BV421 Rat IgG2b, &amp;kappa; Isotype Control RUO</td><td>BD Biosciences</td><td>562603</td><td></td></tr><tr><td>Rabbit anti-mouse TMEM119 (clone28 - 3)</td><td>Abcam</td><td>ab209064</td><td></td></tr><tr><td>AlexaFluor 647 Donkey anti-rabbit IgG</td><td>Life Technologies</td><td>A31573</td><td></td></tr><tr><td>Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Mouse Fc Block</td></tr><tr><td>Fixable Dead Cell Stain Kits</td><td>Invitrogen</td><td>L34969</td><td></td></tr><tr><td>Mouse CCR2 APC-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5538A</td><td></td></tr><tr><td>Rat IgG2B APC-conjugated Isotype Control</td><td>R&amp;amp;D</td><td>IC013A</td><td></td></tr><tr><td>Mouse CX3CR1 PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5825P</td><td></td></tr><tr><td>Goat IgG PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>IC108P</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Cell analyzer</td><td>BD Biosciences</td><td>BD FACSVERSE</td><td></td></tr><tr><td>Data Analysis Software</td><td>FlowJo LLC</td><td>FlowJo</td><td></td></tr><tr><td>Fine scissors</td><td>F.S.T</td><td>14090-11</td><td></td></tr><tr><td>Standard Pattern Forceps</td><td>F.S.T</td><td>11000-13</td><td></td></tr><tr><td>Mayo Scissors</td><td>F.S.T</td><td>14010-15</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>F.S.T</td><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

23.7K 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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Beyin ve Omurilik Mononükleer Hücreleri İzolasyon Percoll Degradeler

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

İmmünoloji ve Enfeksiyon

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Sitometrisi ile Primer İmmün hücre alt Floresan Nanoparçacık Etkileşimleri Algılama

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Bağışıklık hücreleri tek siyatik sinirleri ve Dorsal kök gangliyon üzerinden tek bir fare kullanarak Akış Sitometresi Analizi

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Nörobilim

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

Mononükleer Fagositlerin Çok Parametreli Akış Sitometrik Analizi için Tüm Fare Gözlerinin Sindirimi

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Akış Sitometrisi Kullanılarak Fare Beyni Histon Modifikasyonu Miktar Tayini

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Yazar Spotlight: Gen İfadesi Düzenleme Araştırmasındaki Gelişmeler

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

İzole Fare Beyni Mikrogliasında İntranükleer Akış Sitometrisi Kullanılarak Global Histon Post Translasyonel Modifikasyonların Miktar Tayini

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

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

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Beyin ve Omurilik Mononükleer Hücreleri İzolasyon Percoll Degradeler

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

Sitometrisi ile Primer İmmün hücre alt Floresan Nanoparçacık Etkileşimleri Algılama

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi

Bağışıklık hücreleri tek siyatik sinirleri ve Dorsal kök gangliyon üzerinden tek bir fare kullanarak Akış Sitometresi Analizi

Mononükleer Fagositlerin Çok Parametreli Akış Sitometrik Analizi için Tüm Fare Gözlerinin Sindirimi

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

İzole Fare Beyni Mikrogliasında İntranükleer Akış Sitometrisi Kullanılarak Global Histon Post Translasyonel Modifikasyonların Miktar Tayini

İzole Fare Beyni Mikrogliasında İntranükleer Akış Sitometrisi Kullanılarak Global Histon Post Translasyonel Modifikasyonların Miktar Tayini