この研究は、Agence Nationale Pour la Recherche（ANR-12-MALZ-0003-02-P2X7RAD）、Association France AlzheimerおよびBpifranceからの助成金によって支援された。私たちの研究室は、Inserm、CNRS、Pierre et Marie-CurieUniversidéと「Investissements d&#39;avenir」ANR-10-IAIHU-06（IHU-A-ICM）の支援を受けています。 CELIS細胞培養コア施設の支援に感謝したいと思います。

ここに記載されている全ての方法は、ICM研究所の機関動物管理利用委員会とダーウィンフランス倫理委員会によって承認されており、プロトコル01407.02の適用を受けています。 1.準備<ol><li>各マウスのために以下のものを組み合わせることにより、1.5mLチューブで消化カクテルを調製する：1mLリン酸緩衝食塩水（PBS）; 13 wunsch / mL（ストック溶液）、最終濃度1.6 wunsch / mLで123μLの消化酵素（材料の表参照）。 100mg / mL（ストック溶液）、最終濃度0.5mg / mLで5μLのDNase I（材料の表参照）を添加する。 </li></ol> 2.灌流および切開<ol><li>致死量のペントバルビタール（400mg / mLで100μL）でマウスを安楽死させる。 </li><li>動物を仰臥位に置き、70％エタノールで体を濡らします。 </li><li>胸骨の窩を露出させるために、腸骨棘のちょうど下の細かいはさみで腹部の皮膚を切るy。 </li><li>横隔膜に切開を入れ、心臓を露出させるために（そして他の器官を切断することを避けるために）尾側から吻側への細かいはさみで胸郭の側面を切断する。左心室（LV）と右心房（RA）を特定する。 </li><li> LVの内側に25Gの針を入れたバタフライカテーテルを挿入します。細かいはさみでRAを切開し、血流の開始時に、20mLのPBSを用いて10mL /分の流速で灌流を開始し、血管から細胞および血液を除去する。正常な灌流は、血液が移動するにつれて肺と肝臓との間のブランチングによって示される。 </li><li>中位のはさみで動物を断頭してください。脊柱の腹側にある腹壁筋肉を切除し、尾根の根元で脊柱を切断することにより、脊柱を細かいハサミで解剖し、体から分離する。 </li><li> PBSと腰椎側に200μLピペットチップを搭載している10mLシリンジを挿入脊柱を洗浄し、頚椎側の脊髄を洗い流す。 注：200μLのピペットチップは、最も広い端部（約1cm）ではさみで切断し、PBSであらかじめ充填した10mLのシリンジにしっかりと挿入します。 </li><li>頭蓋骨の上の肌を除去し、脳を暴露するために鉗子を使用してください。頭蓋骨の基部にはさみ刃を挿入し、矢状縫合に続いて頭蓋を切断する。小さな鉗子をカットに挿入し、頭蓋骨を静かに持ち上げます。マウスの頭部から脳を解剖し、嗅球と小脳を除去する。 </li><li> 1.128 mLの消化カクテルを含むPBS（ステップ1.1から）を含む1.5 mLチューブに脳と脊髄を集める。 </li></ol> 3.細胞の解離<ol><li> 1.5 mLチューブ（ステップ2.9）の脳と脊髄を細かいはさみで1〜2 mm 3に細かく切断します （ 図1 ）。 </li><li>細かくした組織を入れた1.5mLのチューブを30分間インキュベートする37℃、5％CO 2でインキュベートする。 注：このステップで密度勾配を準備します。 </li><li>酵素反応を停止するために1.5 mLチューブに0.5μMEDTA（ストック溶液）20μL、最終濃度10 mMを加えます。 </li><li> 5mLのピペットでピペットで上下に静かに細胞懸濁液を作る。 </li><li> 10mLシリンジのプランジャーを使用して、50mLチューブ内のナイロンメッシュ（70μm孔径）に細胞懸濁液を通過させます。 </li><li> 20mLの5％ウシ胎仔血清（FBS）-PBSでナイロンメッシュを洗浄する。 </li><li>室温（18℃）で7分間遠心分離する（300×g）。上清を吸引して捨て、次のステップに進む。上清を取り除くときは十分に注意してください。ペレットを邪魔しないでください。ペレットは吸引ピペットで簡単に吸引できます。 </li></ol> 4.密度勾配<ol><li>適切な量​​の10×PBSを密度勾配媒体に添加することによって、密度勾配媒体の保存溶液を調製する。 注：1部の10X PBSを9部の密度勾配媒体に加えます。 </li><li> 表1に記載されているように、 1 ％PBSでストック密度勾配媒体を異なる割合で希釈する。 </li><li>ペレット（ステップ3.7から）を4mLの30％密度勾配媒体で再懸濁し、15mLチューブに移す。 </li><li>パスツールピペットを15mLチューブの底に置き、37mLの密度勾配培地4mLをゆっくりと下ろす。 </li><li>上記のように、4mLの70％密度勾配媒体を添加する。 </li><li>勾配を室温（18℃）で40分間（800xg）遠心分離する。中間相が妨げられないように、遠心分離機が最小限のブレーキなしで停止することを確認する。 注：チューブは層状に表示されます。 70％〜37％の密度勾配界面で層化された細胞は、単核免疫細胞（小膠細胞およびマクロファージ小集団）である。上位レベルは細胞破片およびミエリンである（ 図2 ）。 </li><li&gt;転送ピペットを使用して、穏やかにミエリンの層を除去する。 </li><li>清潔な15mLのチューブに、マクロファージ亜集団細胞を含む70〜37％密度勾配間期の​​3-4mLを集める。 </li><li>細胞をPBSで3回洗浄し、全量を15mLとする。細胞を含む密度勾配培地をPBSで少なくとも3回希釈し、ブレーキを「オン」にして7分間遠心する（500 xg、4℃）ことを確認します。 </li><li>細胞ラベリングまたは他の機能アッセイのために1mL PBSで細胞ペレットを再懸濁する。 </li></ol>フローサイトメトリー用細胞標識<ol><li>フローサイトメトリーチューブに細胞を移す（ステップ4.10）。 </li><li>ステップ4.10で得られた各細胞サンプルに1μLの固定可能な死細胞染色試薬を加えます。氷上で30分間インキュベートし、光から保護する。 </li><li>細胞を2 mLのPBSで1回洗浄し、5分間遠心（300 xg、4℃）します。 </li><li>上清を除去し、細胞ペレットを400μLのPBS1％BSA、0.1％アジド。 </li><li> 1μgのCD16 / CD32抗体を100μLの細胞に加えて、Fcレセプターをブロックする。氷上で10-15分間インキュベートし、光から保護する。 </li><li>細胞内染色のための細胞浸透化のために、PBS 1％BSA、0.1％アジドまたはPBS 1％BSA、0.1％アジド、0.25％サポニン中の一次抗体混合物を調製する。 </li><li> 100μLの一次抗体ミックス（2×）を添加し、氷上で30分間インキュベートし、光から保護する。 注：最適な結果を得るためには、実験を行う前にすべての抗体を滴定する必要があります。 </li><li> 2mLのPBSで細胞を洗浄し、5分間遠心分離する（300×g、4℃）。この手順を3回繰り返します。 </li><li>一次抗体がフルオロフォアに直接コンジュゲートしていない場合は、100μLの二次抗体を添加し、氷上で30分間インキュベートし、光から保護する。 </li><li> 4℃で300xgで5分間遠心分離して2mLのPBSで細胞を洗浄する。この手順を3回繰り返します。 </li><li>修正する100μLの1％PFAを含む細胞に、光から保護された室温で15分間インキュベートする。 注意：注意：PFAは有毒です。ヒュームフードで使用し、個人用保護具を着用する。 </li><li> 4℃で300 xgで5分間遠心分離して2 mL PBSで細胞を洗浄する。この手順を3回繰り返します。 </li><li> 200μLのPBSで細胞ペレットを再懸濁し、フローサイトメトリーの取得と分析に進みます。 図3のゲーティング戦略に従います 。 </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>

著者は何も開示していない

ミクログリアおよびMDMは、CNSにおいて異なる機能および表現型を有することが実証されており、したがって、これらのマクロファージ亜集団の同定および分析は、神経疾患9,18,25をよりよく理解するために不可欠である。 2つのマーカー（CD11bおよびCD45）を使用するフローサイトメトリー分析は、各亜集団の間の区別を可能にする（ 図3C ）。この戦略は、MDMにつ?...

ミクログリアは、中枢神経系（CNS）の実質組織に存在するマクロファージである。それらは2つの重要な機能的役割を果たします：免疫防御およびCNSホメオスタシスの維持。骨髄の造血幹細胞から絶えず更新されているMDMとは対照的に、ミクログリア細胞は、胚発生の間に脳に定着した卵黄嚢（YS）に由来する原始造血前駆細胞から分化する1,2,3 。げっ歯類では、転写因子Mybは、骨髄由来の単球およびマクロファージのすべての発生に重要な役割を果たすが、YS由来のミクログリアについては、この因子は不可欠であり、分化は転写因子PU.14に依存する。 健康な中枢神経系において、ミクログリアは、絶えず彼らの環境をスクリーニングする動的細胞である侵入する病原体または組織の損傷のための巣作りおよび調査5 。そのようなシグナルの検出は、傷害を解決するための経路を開始する。ミクログリアは、急速に形態を変化させてアメイボイドに転換し、続いて食作用および炎症性サイトカインなどの様々なメディエーターの貪食および放出が起こる。したがって、それらの微小環境に依存して、活性化ミクログリアは、異なるプライミング状態のスペクトルを獲得することができる6 。 ミクログリアは、多くの神経障害の発症および進行に深く影響する。アルツハイマー病（AD） 7 、筋萎縮性側索硬化症（ALS） 8 、多発性硬化症（MS） 9またはパーキンソン病（PD） 10のげっ歯類モデルにおいて、ミクログリアは有害な神経毒性を誘発するか、神経保護的な様式で、これは依存性である特定の疾患、病期、およびその病気が全身性免疫コンパートメント7,8,9,10,11の影響を受けているかどうかを判定した。上記の疾患において観察されるCNS病変の大部分は、実質性小膠細胞だけでなく、血管周囲および髄膜マクロファージならびにCNS浸潤性MDMを含む骨髄系細胞の異種集団を含む。これらの細胞型は、傷害および修復に関連する病態生理学的機構に示差的に寄与する可能性がある7,12,13,14,15。これらの疾患モデルを研究している研究者の現在の課題は、末梢単球およびマクロファージがCNSに浸潤するかどうかを確かめることであり、そうであれば、distこれらの細胞から常在性小膠細胞を摘出する。実際、小膠細胞は非常に塑性である。それらが活性化されると、ミクログリアは、通常、末梢単球およびマクロファージによって発現されるマーカーを再発現する。従って、この問題は、常在ミクログリアを浸潤単球およびマクロファージから区別することができるマーカーを同定することに依存する。 免疫組織学的適用による脳切片上のこれらの集団の識別は、特異的抗体の欠如のために制限される。しかし、フローサイトメトリー分析は、いくつかのマーカーの発現を評価し、細胞集団（例えば、リンパ球、マクロファージ/ MDM CD11b + CD45 high 、およびミクログリアCD11b + CD45 med ）ならびに細胞亜集団16 、 17,18 。このプロトコルは、最適化された酵素組織解離および密度勾配遠心分離を用いることによって神経疾患モデルにおいてマウスCNS由来の単核細胞を単離すること;ならびにフローサイトメトリーを用いてCNS中のミクログリアおよびMDM集団を識別するための方法を提供する。 別のアプローチは、特定の抗体19,20,21にコンジュゲートした磁気ビーズを使用することによってミエリンを除去し、細胞を精製することである。抗ミエリン磁気ビーズを使用するミエリン除去は、より高価であり、単離された細胞22の生存率および収量に影響を及ぼす。この段階およびミクログリアの以下の免疫磁気分離は、特定の免疫細胞集団のさらなる研究を制限する21,22 。 これらの手順は、疾患発症におけるマクロファージ亜集団を研究する簡単な方法を提供し、マクロファージの表現型および活性化状態における薬物効果または遺伝子修飾を決定する。

<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

数多くの研究が、中枢神経系（CNS）病変における免疫細胞、特にマクロファージの役割を実証している。 CNSには、（i）CNSの常在マクロファージであり、胚形成中に卵黄嚢前駆体に由来するミクログリア、および（ii）浸潤し得る単球由来マクロファージ（MDM）の2つの主要なマクロファージ集団が、疾患中のCNSは骨髄前駆細胞に由来する。各マクロファージ亜集団の役割は、研究されている病理によって異なる。さらに、これらのマクロファージ亜集団に使用される組織学的マーカーまたは識別基準にコンセンサスはない。しかし、フローサイトメトリーによるCD11bおよびCD45マーカーの発現プロファイルの分析は、我々がミクログリア（CD11b + CD45 med ）をMDM（CD11b + CD45 high ）から区別することを可能にする。このプロトコルでは、密度勾配遠心分離フローサイトメトリー分析を用いて、これらのCNSマクロファージ亜集団を特徴づけ、最近発表されたこれらの細胞によって発現されるいくつかの興味深いマーカーを研究することができる。従って、この技術は、神経疾患のマウスモデルにおけるマクロファージの役割をさらに理解することができ、これらの細胞に対する薬物効果を評価するためにも使用することができる。

密度勾配遠心分離および抗体染色の後、細胞をフローサイトメーターで取得し、以下のような形態学的ゲーティング戦略を用いて分析した。単一の細胞を二重線から区別するために、ドットプロット前方散乱領域（FSC-A） 対前方散乱高（FSC-H）で第1ゲートを定義した（ 図3A ）。次いで、相対細胞サイズおよび細胞粒度に基づいて、細胞破...

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

このプロトコールは、成体マウス中枢神経系におけるマクロファージ亜集団のフローサイトメトリーによる分析を提供し、これらの細胞によって発現される複数のマーカーの研究に有用である。

フローサイトメトリーによる中枢神経系からのミクログリアおよび単球由来マクロファージの解析

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 ビュー.  Inserm U 1227, CNRS UMR 7225. このプロトコールは、成体マウス中枢神経系におけるマクロファージ亜集団のフローサイトメトリーによる分析を提供し、これらの細胞によって発現される複数のマーカーの研究に有用である。 

ビデオ: 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.

新生児と大人の中枢神経系から培養ミクログリア

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

免疫・感染学

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.

マクロファージコロニー刺激因子分化したヒト単球由来マクロファージの文化

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.

イメージングフローサイトメトリーによるヒト単球由来樹状細胞の特徴付け：二つの単球の分離プロトコルの比較

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.

フローサイトメトリーによるラットの腎臓からのマクロファージの表現型の特徴

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.

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

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.

離とマウスとヒトの肺胞マクロファージの体外培養

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).

全血によって人間の Monocyte のサブセットの解析フローのフローサイトメトリー解析

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.

実験的自己免疫性脳脊髄炎における中枢神経系におけるリンパ球浸潤のフロー細胞量分析

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.

免疫細胞の下流分析のための中枢神経系組織および関連する髄膜の単離

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.

フローサイトメトリー解析のためのマウス腎臓レジデントCD8+T 細胞の分離

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 ...