Name: isolation and in vitro culture of murine and human alveolar macrophages
Uploaded: 2018-04-20T14:00:00
Description: Watch this Scientific Journal Video about Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages at JoVE.com

原稿を編集の支援ありがとうクレア プレンダーガスト。DKN はサポートされて研究でフリン財団から (#2095) を付与し、TM は国立衛生研究所 (R01HL056643 と R01HL092514) からの補助金によってサポートされています。DKN 手法を開発、研究をデザイン、そして書いた原稿;動物実験と臨床サンプル調達支援 OMSB 支援フローサイトメトリー解析・細胞のソーティング。TM は研究を監督し、原稿の見直し。

ここで説明したすべてのメソッドは、機関動物ケアおよび使用委員会 (IACUC) セントジョセフ病院と医療センター制度レビュー委員会 (IRB) によって承認されています。
1. マウスの気管支肺胞洗浄 (BAL) 液から AMs の分離
<ol><li>ケタミン (87.5 mg/kg 体重) とキシラジン (12.5 mg/kg 体重) 腹腔内注射を介してカクテル 8 週齢 c57bl/6 マウスを anaesthetize します。マウスに達する反射の...

<ol>
	<li>Westphalen, K., 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=Sessile+alveolar+macrophages+communicate+with+alveolar+epithelium+to+modulate+immunity.">Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity.</a> Nature. 506 (7489), 503-506 (2014).</li><li>Guilliams, 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=Alveolar+macrophages+develop+from+fetal+monocytes+that+differentiate+into+long-lived+cells+in+the+first+week+of+life+via+GM-CSF.">Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF.</a> J Exp Med. 210 (10), 1977-1992 (2013).</li><li>Cardani, A., Boulton, A., Kim, T. S., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+prevent+lethal+influenza+pneumonia+by+inhibiting+infection+of+type-1+alveolar+epithelial+cells.">Alveolar macrophages prevent lethal influenza pneumonia by inhibiting infection of type-1 alveolar epithelial cells.</a> PLoS Pathog. 13 (1), e1006140 (2017).</li><li>Ghoneim, H. E., Thomas, P. G., McCullers, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+alveolar+macrophages+during+influenza+infection+facilitates+bacterial+superinfections.">Depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections.</a> J Immunol. 191 (3), 1250-1259 (2013).</li><li>MacLean, J. 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=Sequestration+of+inhaled+particulate+antigens+by+lung+phagocytes.+A+mechanism+for+the+effective+inhibition+of+pulmonary+cell-mediated+immunity.">Sequestration of inhaled particulate antigens by lung phagocytes. A mechanism for the effective inhibition of pulmonary cell-mediated immunity.</a> Am J Pathol. 148 (2), 657-666 (1996).</li><li>Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+alveolar+macrophages+by+clodronate-liposomes+aggravates+ischemia-reperfusion+injury+of+the+lung.">Depletion of alveolar macrophages by clodronate-liposomes aggravates ischemia-reperfusion injury of the lung.</a> J Heart Lung Transplant. 24 (1), 38-45 (2005).</li><li>Schneider, 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=Alveolar+macrophages+are+essential+for+protection+from+respiratory+failure+and+associated+morbidity+following+influenza+virus+infection.">Alveolar macrophages are essential for protection from respiratory failure and associated morbidity following influenza virus infection.</a> PLoS Pathog. 10 (4), e1004053 (2014).</li><li>Pribul, P. K., 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=Alveolar+macrophages+are+a+major+determinant+of+early+responses+to+viral+lung+infection+but+do+not+influence+subsequent+disease+development.">Alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development.</a> J Virol. 82 (9), 4441-4448 (2008).</li><li>Macdonald, 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=Harnessing+alveolar+macrophages+for+sustained+mucosal+T-cell+recall+confers+long-term+protection+to+mice+against+lethal+influenza+challenge+without+clinical+disease.">Harnessing alveolar macrophages for sustained mucosal T-cell recall confers long-term protection to mice against lethal influenza challenge without clinical disease.</a> Mucosal Immunol. 7 (1), 89-100 (2014).</li><li>Benoit, A., Huang, Y., Proctor, J., Rowden, G., Anderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alveolar+macrophage+depletion+on+liposomal+vaccine+protection+against+respiratory+syncytial+virus+(RSV).">Effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (RSV).</a> Clin Exp Immunol. 145 (1), 147-154 (2006).</li><li>Nayak, D. K., 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=Long-term+persistence+of+donor+alveolar+macrophages+in+human+lung+transplant+recipients+that+influences+donor+specific+immune+responses.">Long-term persistence of donor alveolar macrophages in human lung transplant recipients that influences donor specific immune responses.</a> Am J Transplant. 16 (8), 2300-2311 (2016).</li><li>Sekine, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+passenger+leukocytes+in+allograft+rejection:+effect+of+depletion+of+donor+alveolar+macrophages+on+the+local+production+of+TNF-alpha,+T+helper+1/T+helper+2+cytokines,+IgG+subclasses,+and+pathology+in+a+rat+model+of+lung+transplantation.">Role of passenger leukocytes in allograft rejection: effect of depletion of donor alveolar macrophages on the local production of TNF-alpha, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation.</a> J Immunol. 159 (8), 4084-4093 (1997).</li><li>Borie, 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=Pulmonary+alveolar+proteinosis.">Pulmonary alveolar proteinosis.</a> Eur Respir Rev. 20 (120), 98-107 (2011).</li><li>Greenhill, S. R., Kotton, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+alveolar+proteinosis:+a+bench-to-bedside+story+of+granulocyte-macrophage+colony-stimulating+factor+dysfunction.">Pulmonary alveolar proteinosis: a bench-to-bedside story of granulocyte-macrophage colony-stimulating factor dysfunction.</a> Chest. 136 (2), 571-577 (2009).</li><li>Happle, 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=Pulmonary+transplantation+of+macrophage+progenitors+as+effective+and+long-lasting+therapy+for+hereditary+pulmonary+alveolar+proteinosis.">Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.</a> Sci Transl Med. 6 (250), 250ra113 (2014).</li><li>Suzuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+macrophage+transplantation+therapy.">Pulmonary macrophage transplantation therapy.</a> Nature. 514 (7523), 450-454 (2014).</li><li>Hashimoto, 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=Tissue-resident+macrophages+self-maintain+locally+throughout+adult+life+with+minimal+contribution+from+circulating+monocytes.">Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.</a> Immunity. 38 (4), 792-804 (2013).</li><li>Murphy, J., Summer, R., Wilson, A. A., Kotton, D. N., Fine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prolonged+life-span+of+alveolar+macrophages.">The prolonged life-span of alveolar macrophages.</a> Am J Respir Cell Mol Biol. 38 (4), 380-385 (2008).</li><li>Maus, U. 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=Resident+alveolar+macrophages+are+replaced+by+recruited+monocytes+in+response+to+endotoxin-induced+lung+inflammation.">Resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation.</a> Am J Respir Cell Mol Biol. 35 (2), 227-235 (2006).</li><li>Perdiguero, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Bitterman, P. B., Saltzman, L. E., Adelberg, S., Ferrans, V. J., Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophage+replication.+One+mechanism+for+the+expansion+of+the+mononuclear+phagocyte+population+in+the+chronically+inflamed+lung.">Alveolar macrophage replication. One mechanism for the expansion of the mononuclear phagocyte population in the chronically inflamed lung.</a> J Clin Invest. 74 (2), 460-469 (1984).</li><li>Misharin, A. V., 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=Monocyte-derived+alveolar+macrophages+drive+lung+fibrosis+and+persist+in+the+lung+over+the+life+span.">Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.</a> J Exp Med. , (2017).</li><li>Zheng, Z., 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=Donor+pulmonary+intravascular+nonclassical+monocytes+recruit+recipient+neutrophils+and+mediate+primary+lung+allograft+dysfunction.">Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction.</a> Sci Transl Med. 9 (394), (2017).</li><li>Nayak, D. K., 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=Zbtb7a+induction+in+alveolar+macrophages+is+implicated+in+anti-HLA-mediated+lung+allograft+rejection.">Zbtb7a induction in alveolar macrophages is implicated in anti-HLA-mediated lung allograft rejection.</a> Sci Transl Med. 9 (398), (2017).</li><li>Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R., Perlman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macrophages+and+dendritic+cell+subsets+in+the+mouse+lung.">Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung.</a> Am J Respir Cell Mol Biol. 49 (4), 503-510 (2013).</li><li>Kopf, M., Schneider, C., Nobs, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+function+of+lung-resident+macrophages+and+dendritic+cells.">The development and function of lung-resident macrophages and dendritic cells.</a> Nat Immunol. 16 (1), 36-44 (2015).</li><li>Eguiluz-Gracia, I., 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=Long-term+persistence+of+human+donor+alveolar+macrophages+in+lung+transplant+recipients.">Long-term persistence of human donor alveolar macrophages in lung transplant recipients.</a> Thorax. 71 (11), 1006-1011 (2016).</li><li>Yu, Y. 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=Flow+cytometric+analysis+of+myeloid+cells+in+human+blood,+bronchoalveolar+lavage,+and+lung+tissues.">Flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues.</a> Am J Respir Cell Mol Biol. , (2015).</li></ol>

AMs は、肺に生じる出生時と26全体の寿命を永続的な作成時間の長いリビング肺居住者マクロファージです。肺生理学7と病理12とその潜在的な肺の免疫24の予測における役割を認識しています。AMs 肺11,27で長期的な存在感があるので、活性化と免疫11,<sup...

肺微小環境は精巧な空気の導管と血管を一意に複雑な生態系です。吸入空気は、血液-空気ガス交換が発生する、肺胞に達する前に気管と気管支、細気管支の多数の枝を通してを移動します。大気と直接相互作用は、呼吸の表面は浮遊粒子状物質や汚染物質の可能性のある有害な効果から保護を必要とします。物理的、化学的、免疫学的障壁の数は、肺を保護します。特に、食細胞呼吸面での展開は、重要な最初の行の防衛システムを提供しています。肺胞マクロファージ (AMs) 肺居住者食細胞の一種であるし、彼らは肺マクロファージ プールの大半を構成します。その名の通り、AMs は主に肺胞腔に局在しているし、常に周囲の雰囲気をサンプルし、歯槽の上皮1通信付着細胞として発生します。肺胞腔内の食細胞の 95% 以上は、定常状態の肺、AMs2、その組成は、炎症、感染症、または汚染物質への慢性露出により変わる可能性があります。
AMs は、全身の重要性のおよび/または肺に対してローカルにすることができる機能の広い範囲に参加します。例えば、AMs は肺の最適な機能と開発に不可欠です。免疫の監視;侵入する病原体と吸入粒子3,4,5,6,7細胞の残骸のクリアランス。AMs の対象の枯渇は、呼吸器系ウイルスや細菌4,8のクリアランスを阻害する知られています。食細胞と肺の恒常性の最初の行の擁護者としての役割のほか AMs 知られている T を引き出すことで抗原提示細胞細胞免疫9、として機能する鼻腔内ワクチン10の効果を増強し、肺移植11,12後肺制限自己免疫に影響を与えます。午前の機能の欠乏は、遺伝子の突然変異、悪性腫瘍や肺界面活性剤13,14のクリアランスを損なう感染から生じる条件 (PAP)、肺胞蛋白症にリンクされています。AMs の移植は今 PAP 15,16の治療のための治療法として検討されています。
AMs は、胚形成の間に由来して循環白血球2,17に置き換えることがなく一生肺に固執するに知られています。午前売り上げ高のさまざまなレベルがインフルエンザ ウイルス4、破壊的照射18、内毒素への暴露によって感染を含む特定の臨床状況で報告されている午前売り上げ高は恒常性の肺に、19、そして高齢者20。AMs は、自己を介して悪性増殖17,21, 更新すると考えられているが、いくつかの最近の研究は、単球が血管肺マクロファージ22,23の下の人口に上昇を与えることができることを主張これらの新たに変えられた肺のマクロファージの機能がの実験条件はまだ肺疾患定義しなければなりません。肺炎症シグナルと免疫制御機械間の平衡を維持しようとすると、潜在的に興味深い地域がさらに午前活性化のコンテキストで刺激の閾値を理解することです。
免疫制御の損失をもたらす生理学的または病理学的変化は、さまざまな臨床設定 (例えば、呼吸器感染症、炎症性肺疾患、肺線維化疾患) の評価に重要です。それにもかかわらず、AMs はますますインジケーターまたは肺健康11,24のも決定要因として認識されます。収穫、特性評価、および人間および前臨床マウスモデルから維持する AMs 統一プロトコルは現在、ありません。午前前駆体と表現型のコンセンサスの欠如と詳細な方法の不在は、肺の健康および病気の午前の解読の役割の主要な障害をされていた。次のプロトコルでは、決定的な識別、分離、および体外で文化大きく午前動作の理解を進めるし、AM 向けの診断および治療の調査を促進する戦略を提供しています。

<table border="0" cellpadding="0" cellspacing="0" width="910">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Non-enzymatic cell dissociating solution</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td style="width:220px;">C5789</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube Vet Ointment</td>
			<td style="width:223px;">Dechra</td>
			<td>620300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G Catheter&#160;</td>
			<td style="width:223px;">Terumo Medical Products</td>
			<td>SR-OX2225CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Non-absorbable silk braided suture&#160;</td>
			<td style="width:223px;">Kent Scientific</td>
			<td>SUT-15-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Fc block&#160;</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysis buffer (PureLink RNA Kit)</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>12183018A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>M6250&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAria II cell sorter&#160;</td>
			<td style="width:223px;">BD Biosciences</td>
			<td style="width:220px;">644832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160; (Ketathesia)</td>
			<td style="width:223px;">Henry Schein</td>
			<td style="width:220px;">56344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160; (AnaSed)</td>
			<td style="width:223px;">Akorn</td>
			<td style="width:220px;">139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">10-040-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liberase TL&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td style="width:220px;">5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>AMPD1-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100&#956;m cell strainer&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Human Fc block</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">EDTA</td>
			<td style="width:223px;">Corning</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Trypan Blue Solution</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">L-929 cell line</td>
			<td>American Type Culture Collection</td>
			<td>ATCC,&#160;CCL-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate</td>
			<td style="width:223px;">Corning</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T25 Tissue culture flask</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm culture dish&#160;</td>
			<td style="width:223px;">Millipore-Sigma</td>
			<td>CLS3261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">15 mL Conical tube&#160;</td>
			<td style="width:223px;">Corning</td>
			<td style="width:220px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">50 mL Conical tube&#160;</td>
			<td style="width:223px;">Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRFortessa cell analyzer</td>
			<td>BD Biosciences</td>
			<td>657669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">FlowJo</td>
			<td style="width:223px;">FlowJo</td>
			<td>v10.4</td>
			<td>Analysis Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD45 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>147709</td>
			<td>Clone I3/2.3, FITC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11b (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>101228</td>
			<td>Clone M1/70, PerCP/Cy5.5 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11c (Mouse)</td>
			<td>BD Biosciences</td>
			<td>565452</td>
			<td>Clone N418, BV 421 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-I-Ab (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>116420</td>
			<td>Clone AF6-120.1, PE/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Siglec-F (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>562757</td>
			<td>Clone E50-2440, PE-CF594 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Siglec-H (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>129605</td>
			<td>Clone 551, PE conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-F4/80 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>123118</td>
			<td>Clone BM8, APC/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-Ly-6C (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>128035</td>
			<td>Clone HK1.4, BV605 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD64 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>139311</td>
			<td>Clone X54-5/7.1, BV711 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD24 (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>563115</td>
			<td>Clone M1/69, BV510 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD103 (Mouse)</td>
			<td style="width:223px;">BD Biosciences</td>
			<td>745305</td>
			<td>Clone OX-62, BV650 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD317 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>127015</td>
			<td>Clone 927, APC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CXCR1 (Mouse)</td>
			<td style="width:223px;">Biolegend</td>
			<td>149029</td>
			<td>Clone SA011F11, BV785 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD45 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>304017</td>
			<td>Clone HI30, AF488 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD11b (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>101216</td>
			<td>Clone M1/70, PE/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-HLA-DR (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>307618</td>
			<td>Clone L243, APC/Cy7 conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD169 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>346008</td>
			<td>Clone 7-239, APC conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD206 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>321106</td>
			<td>Clone 15-2, PE conjugated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Anti-CD163 (Human)</td>
			<td style="width:223px;">Biolegend</td>
			<td>333612</td>
			<td>Clone GHI/61, BV421 conjugated</td>
		</tr>
	</tbody>
</table>

isolation and in vitro culture of murine and human alveolar macrophages

肺胞マクロファージは、肺居住者、最終分化マクロファージ出生前原産です。肺胞マクロファージは、寿命が長く、肺開発機能とその肺局所応答感染症や炎症に重要な役割でユニークです。日には、識別、分離、および人間およびマウスから肺胞マクロファージの処理の統一的手法は存在しません。このようなメソッドは、さまざまな実験的設定のこれらの重要な自然免疫系の細胞に関する研究に必要です。あらゆる研究室で簡単に採用できる、ここで説明する方法は、気管支肺胞洗浄液や肺組織から肺胞マクロファージを採取し、体外を維持する簡素化されたアプローチです。肺胞マクロファージは、肺胞に付着性のセルとして主に発生する、ためこのメソッドの焦点は収穫と同定する前にそれらを外れです。肺は高度に血管の臓器、骨髄性とリンパ性の起源の様々 な細胞タイプに生息する、対話、および肺微小環境によって影響を受けています。ここで説明されている表面のマーカーのセットを使用して、研究者はことができます簡単かつ明確に他の白血球から肺胞マクロファージを区別し、下位アプリケーションにそれらを浄化します。ここを開発した培養法両方の人間をサポートするマウスの体外発育の肺胞マクロファージと分子・細胞の研究と互換性が。

マウス AMs を識別するためにフローのフローサイトメトリーによるアプローチは、図 1に示すです。これは、表面マーカー他肺居住者又は肺に浸潤食から AMs を区別するに必要な最小セットの分析が含まれています。差分解析は、間質性マクロファージ、樹状細胞、好中球、単球、肺に発生する単球由来の肺マクロファージから AMs を識別するた?...

Watch this Scientific Journal Video about Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages at JoVE.com

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

離とマウスとヒトの肺胞マクロファージの体外培養

この通信は、人間から肺胞大食細胞の分離と培養の方法論や実験用マウスのモデルについて説明します。

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

Research

JoVE Journal

Immunology and Infection

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

移行のヒトTリンパ球の単離、培養および解析インビトロ</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

ダブル勾配遠心、テフロンコーティングされた細胞培養バッグ中のマクロファージへの分化によるヒト単球の単離

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

共焦点顕微鏡による絶縁型肺胞マクロファージの抗真菌活性を評価する

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Toll様受容体刺激時の遺伝子発現解析を行うためにマウス腹腔マクロファージの単離

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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

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 ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

ひと単球走化性の in VitroおよびIn Vivoマウス リンパ球の定量化

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...