我们感谢克莱尔协助编辑手稿。DKN 由克里斯弗林基金会的研究补助金 (#2095) 支持, TM 得到国家卫生研究院 (R01HL056643 和 R01HL092514) 赠款的支持。DKN 开发了方法, 设计了研究并撰写了手稿;协助动物研究和临床样品采购;协助进行流细胞分析和细胞分选;TM 监督研究和审阅手稿。

这里所描述的所有方法都已被 IACUC 医院和医疗中心的机构动物护理和使用委员会 (IRB) 和机构审查委员会批准。
1. 从小鼠支气管肺泡灌洗 (球) 液中分离 AMs
<ol><li>麻醉一个八周大的 C57BL/6 老鼠与氯胺酮 (87.5 毫克/千克体重) 和甲苯噻嗪 (12.5 毫克/千克体重) 鸡尾酒通过腹腔注射。当老鼠获得手术麻醉, 失去反射和肌肉松弛。</li>
	<li>将鼠标放在解剖表面, 侧面朝上。应用眼科兽医软膏, 防止脱水, 用70% 异丙醇饱和无菌的酒精准备垫擦拭小鼠的整个腹面, 以进行消毒。</li>
	<li>装上所有四条腿克制的老鼠。</li>
	<li>用微解剖工具轻轻地打开腹腔。必须注意尽量打开尽可能多的区域, 而不损害内脏器官或制造悬垂组织。</li>
	<li>通过纵隔缓慢切开胸腔, 轻轻地打开胸廓。理想情况下, 肋骨笼可以从一侧到一侧切除。在打开胸腔时, 必须格外小心, 不要伤害肺部的胸膜。</li>
	<li>定位下腔静脉, 并进行切口出血鼠标。使用吸收棉垫吸收血液。</li>
	<li>将10毫升冰寒磷酸盐缓冲盐水 (PBS) 注入右心室 (使用10毫升注射器和25G 针) 冲洗循环血细胞。用吸收棉垫吸收血液流动。一旦完全灌注, 肺部将出现脱皮, 然后准备冲洗。</li>
	<li>轻轻切开颈部的皮肤和肌肉以暴露气道。小心地切除相邻的肌肉、软骨和脂肪组织, 而不损害气管。</li>
	<li>在喉后部的气管上做一个小切口 (< 2 毫米)。这个切口应该只是足够插入导管进入, 而气管仍然连接到喉。插入一个1英寸的22G 导管, 没有针进入气管向肺部。用丝编织缝线 (4-0; 不可吸收) 用方形结固定导管。 注: 导管需要根据鼠标大小进行修整。在灌洗过程中, 极短或长的导管往往会泄漏和/或撕裂气道。导管长度应如此, 当充分插入尖端保持良好的气管内, 而不是到达主支气管。</li>
	<li>将1毫升注射器装满冰冷的球缓冲器 (Ca2 +和 Mg2 +免费 PBS + 1 毫米乙二胺四乙酸, EDTA) 到导管中, 慢慢将缓冲液注入肺部。这会使肺部膨胀。将注射器固定在导管上五秒钟, 然后轻轻拉动活塞, 然后吸入灌洗液。这会使肺部降温。在冰上放置15毫升锥形管中的液体。</li>
	<li>重复步骤1.10 九次, 并池灌洗液。不要超过1毫升的缓冲每冲洗, 因为这可能超过肺容量, 并导致其破裂。</li>
	<li>通过 IACUC 批准的协议, 将鼠标与 CO2过量弄死。</li>
	<li>离心机10毫升的液体在 250 x g 4 °c 10 分钟。细胞颗粒含有球细胞。 警告:颗粒可能很小, 所以在吸上清液时要小心。</li>
	<li>并用重悬在 100 ul 的流动/排序缓冲 (PBS + 2% 的热灭活胎牛血清 (血清) + 1 毫米 EDTA + 25 毫米 N-2-Hydroxyethylpiperazine-N'-2-ethanesulphonic 酸 (HEPES) 中的球细胞。</li>
	<li>通过添加小鼠 fc 块 (克隆 2.4G2, 10 微克/毫升), 并在冰上孵化20分钟, 阻止非特异性抗体绑定到 Fc 受体。</li>
	<li>执行抗体染色与荧光减一 (FMO) 和单污点控制11,25。用细胞分析仪 (参见材料表) 评估球细胞, 然后用适当的分析软件进行单细胞流式细胞分析 (见材料表)。</li>
	<li>当 ams 用于体外区域性或体内细胞传输时, 将 ams 由荧光活化细胞分拣器 (参见材料表) 直接导入1毫升的鼠标 AM 培养基中。或者, 将 AMs 分类为1毫升裂解缓冲器 (参见材料表), 辅以 10 ul/毫升β-巯基乙醇进行 RNA 提取。</li>
</ol>2. 从小鼠肺、单细胞悬浮液中分离 AMs
<ol><li>麻醉一个八周大的 C57BL/6 老鼠与氯胺酮 (87.5 毫克/千克体重) 和甲苯噻嗪 (12.5 毫克/千克体重) 鸡尾酒通过腹腔注射。当老鼠获得手术麻醉, 失去反射和肌肉松弛。</li>
	<li>将鼠标放在解剖表面, 侧面朝上。应用眼科兽医软膏, 防止脱水, 用70% 异丙醇饱和的无菌酒精垫擦拭小鼠的整个腹面, 以进行消毒。</li>
	<li>装上所有四条腿克制的老鼠。</li>
	<li>用微解剖工具轻轻地打开腹腔。必须注意尽量打开尽可能多的区域, 而不损害内脏器官或制造悬垂组织。</li>
	<li>通过纵隔缓慢切开胸腔, 轻轻地打开胸廓。理想情况下, 肋骨笼可以从一侧到一侧切除。在打开胸腔时, 必须格外小心, 不要伤害肺部的胸膜。</li>
	<li>定位下腔静脉, 并进行切口出血鼠标。使用吸收棉垫吸收血液。</li>
	<li>将10毫升冰冷 PBS 注入右心室 (使用10毫升注射器和25G 针) 冲洗循环血细胞。一旦完全灌注, 肺部将出现脱皮, 并准备收割。</li>
	<li>通过将气管、血管和韧带切开到15毫升圆锥管中, 用5毫升的冷 Dulbecco 修饰鹰的培养基 (DMEM) 来解剖肺部。保持它在冰上, 直到下一步。</li>
	<li>通过 IACUC 批准的协议, 将鼠标与 CO2过量弄死。</li>
	<li>将灌注后的肺部转移到无菌和无热原体的60毫米培养皿中, 3 毫升 DMEM。借助解剖钳逗出呼吸道和其他硬非肺组织材料, 如果存在。用手术刀将肺组织切碎, < 1 毫米大小。</li>
	<li>添加300µg/毫升 Liberase TL 和 5 U/毫升 DNase I, 混合吹打和孵化在 37° C 在孵化器25分钟。10分钟后, 用吹打轻轻混合一次。</li>
	<li>通过100µm 细胞过滤器安装在50毫升圆锥管, 通过分离的肺部。使用从1毫升注射器的柱塞后, 把细胞团簇在过滤器上。清洗过滤器与20毫升洗涤缓冲 (PBS + 2% 热灭活血清 + 2 毫米 EDTA)。</li>
	<li>离心机在 500 x g 在4°c 5 分钟。丢弃上清液, 并用重悬20毫升洗涤缓冲器中的细胞颗粒。</li>
	<li>重复步骤2.12 和2.13 两次, 每次更换过滤器和管。在洗涤缓冲器中预浸泡过滤器超过五分钟, 增加了 AM 产量。通过将 100 ul 流/分选缓冲器加入细胞颗粒, 制备单细胞悬浮液。</li>
	<li>通过添加小鼠 fc 块 (克隆 2.4G2, 10 微克/毫升) 和孵化20分钟冰, 阻止非特异性抗体结合到 Fc 受体。</li>
	<li>使用 FMO 和单污点控件执行抗体染色11,25。用细胞分析仪 (参见材料表) 评估肺细胞, 然后用分析软件进行单细胞流式细胞仪分析。</li>
	<li>当 ams 用于体外区域性或体内细胞传输时, 将 ams 按单元格排序器 (参见材料表) 直接转换为1毫升的鼠标 AM 培养基。或者, 将 AMs 分类为1毫升裂解缓冲器, 辅以 10 ul/毫升β-巯基乙醇 RNA 萃取。</li>
</ol>3. 从人的体液中分离 AMs
<ol><li>安排将人的体液从诊所转移到实验室4摄氏度。</li>
	<li>离心的球液 (10-50 毫升) 在 250 x g 在4°c 10 分钟。细胞颗粒含有球细胞11。</li>
	<li>用20毫升洗涤缓冲剂 (PBS + 2% 热灭活的血清 + 2 毫米 EDTA) 和离心机在 250 x g 处用4摄氏度清洗颗粒10分钟。</li>
	<li>并用重悬细胞在 100 ul 的流/排序缓冲 (PBS + 2% 热灭活血清 + 1 毫米 EDTA + 25 毫米 HEPES)。</li>
	<li>通过添加人 fc 块 (克隆 Fc1.3070, 25 微克/毫升), 并在冰上孵化20分钟, 阻止非特异性抗体结合到 Fc 受体。</li>
	<li>使用 FMO 和单污点控件执行抗体染色11,24。用细胞分析仪 (参见材料表) 对球细胞进行评估, 然后用分析软件进行单细胞流式细胞仪分析。</li>
	<li>将 AMs 按单元格排序器 (参见材料表) 直接添加到1毫升人类培养基或1毫升裂解缓冲器中, 辅以 10 ul/毫升β-巯基乙醇。</li>
</ol>4. AMs 的体外培养
<ol><li>在细胞分类之后, 通过离心在 250 x g 的4摄氏度处获得10分钟的细胞。</li>
	<li>并用重悬人类 AMs 在 1毫升人类AM 培养基 [罗斯威尔公园纪念研究所培养基 (RPMI) 1640 补充10% 血清, 20% L-929 培养上清 (作为巨噬细胞集落刺激因子 (M CSF), 100 U/毫升最终浓度的来源),1毫米丙酮酸钠, 10 毫米 N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic 酸 (HEPES), 1x 青霉素/链霉素 (可选)]。同样, 并用重悬小鼠 AMs 在 1毫升鼠标AM 培养基 [DMEM 补充10% 的血清, 20% L-929 培养上清 (作为脑脊液, 100 U/毫升最终浓度), 1 毫米丙酮酸钠, 10 毫米 HEPES 和1x 青霉素/链霉素 (可选)].</li>
	<li>用单元格计数器枚举台盼蓝-排除活细胞, 并在 1 x 106/mL 上调整活细胞浓度。 注意: 理想情况下, 多达 5 x 105 AMs 可以与从 8-10 周老 C57BL/6 雄性老鼠收集的球液隔离。小鼠肺消化液的产率是可变的, 可能略小于小鼠的体液。从人的体液中获得的产量取决于球液的体积、灌洗中使用的生理盐水总量以及病人的临床状况。</li>
	<li>根据产量和实验需要, 种子细胞在室内滑动, 培养皿或培养瓶。理想情况下, 种子的 AMs 在 1 x 105/毫升与10毫升培养基在一个 T25 组织培养瓶。</li>
	<li>用 5% CO2大气在37°c 湿润孵化箱中孵化细胞。 注意: AMs 将成长为黏附细胞和非常缓慢增长 (加倍的时间 > 7 天)。</li>
	<li>在播种后的24小时内, AMs 应该是黏附的, 不会被一个温和的改变文化培养基分离。从一端吸取吸入培养基, 并补充新鲜培养基预热至37摄氏度。 注: 细胞现在可以用来评估 AM 生理或刺激的影响。在腔内生长的 AMs 可以方便地沾上适当的抗体, 是显微可视化的理想选择。对于流细胞评价, AMs 是通过非酶细胞游离溶液的孵化而收获的。</li>
	<li>吸入培养基并添加游离溶液, 足以覆盖培养面 (每 T25 瓶大约2毫升), 在37摄氏度孵化 5-10 分钟。不需要刮擦, 不建议用机械的刮伤细胞。轻轻地拍打两侧, 加入 1-2 毫升的流/排序缓冲和轻轻的吸管向上和向下分离大多数黏附细胞。</li>
	<li>在 RNA 研究中, 通过添加裂解缓冲剂, 将细胞直接溶解在培养皿上。</li>
</ol>

<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>

作者没有要申报的利益冲突。

AMs 是长寿的肺常驻巨噬细胞, 在出生时填充肺部, 并持续超过整个生命周期26。他们的作用在肺生理学7和病理学12和他们的潜力预测肺自身免疫24已经被认可。由于 AMs 长期存在于肺部11、27中, 并且由于它们参与了免疫应答的激活和进展11,24</sup...

肺微环境是一个独特的复杂的生态系统, 具有精心设计的空气导管和血管。吸入的空气通过气管和通过许多分支支气管和性细支气管在到达肺泡之前, 那里血液空气气体交换发生。由于与大气层的直接相互作用, 呼吸表面需要保护, 免受空气微粒和污染物的潜在有害影响。一些物理、化学和免疫屏障保护肺部。值得注意的是, 在呼吸表面部署吞噬是一项重要的一线防御系统。肺泡巨噬细胞 (AMs) 是肺吞噬的一种类型, 它们构成了肺巨噬细胞的绝大部分。顾名思义, AMs 主要定位于肺泡腔, 并作为无梗细胞持续取样周围环境, 并与肺泡上皮1进行通信。在稳态肺中, 95% 以上的吞噬在肺泡空间是 AMs2, 其组成可能会改变, 由于炎症, 感染, 或长期接触污染物。
AMs 参与范围广泛的功能, 可能是局部的肺部和/或系统的重要性。例如, AMs 对肺部的发育和最佳功能至关重要;免疫监测;和清除细胞碎片、入侵病原体和吸入微粒3,4,5,6,7。有针对性地耗尽 AMs, 损害呼吸病毒和细菌的清除,4,8。除了作为吞噬和一线防御者的肺稳态, AMs 是已知的功能作为抗原呈现细胞诱导 T 细胞免疫9, potentiating 鼻腔疫苗的功效10和肺移植后肺限制自身免疫的影响11,12。AM 功能不足已与肺肺泡支持下肺泡 (PAP) 相关联, 这是由基因突变、恶性肿瘤或感染损害肺表面活性剂清除率13, 14 的条件造成的.目前正在探索 AMs 移植治疗 PAP 1516的治疗方法。
AMs 是已知的起源于胚胎发生过程中, 并坚持在整个生命中的肺部, 而不被循环白细胞取代2,17。虽然, 在稳态肺中, am 的营业额是无法检测到的, 但在某些临床情况下, 有不同程度的 am 更替, 包括感染流感病毒4、清髓性照射18、接触内毒素19和老年20。AMs 被认为通过低级别的增生17,21进行自我更新, 但最近的一些研究声称单核细胞可以引起血管内肺巨噬细胞的数量22,23下实验条件, 但这些新转换的肺巨噬细胞的功能尚未定义在肺部疾病。此外, 在 AM 活化的背景下, 了解刺激的阈值是一个潜在的有趣的领域, 因为肺试图保持炎症信号和免疫调节机制之间的平衡。
导致免疫调节丧失的生理或病理改变对于评估各种临床环境 (例如,呼吸道感染、炎症性肺病和纤维化肺病) 是很重要的。然而, AMs 越来越被认为是肺健康的指标, 甚至是决定因素,11,24。目前, 尚无统一的协议可用于收割、鉴定和/或维持来自人类和前小鼠模型的 AMs。对 am 前体和表型缺乏共识, 缺少详细的方法是破译 AM 在肺部健康和疾病中作用的主要障碍。下面的协议提供了一个明确的标识、隔离和体外文化策略, 它将极大地促进对 am 行为的理解, 并促进有针对性的诊断和治疗研究。

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

肺泡巨噬细胞是末期分化的, 肺部驻留的巨噬细胞。肺泡巨噬细胞在其漫长的生命中是独一无二的, 它们在肺发育和功能方面的重要作用, 以及它们对感染和炎症的肺部局部反应。迄今为止, 没有统一的方法来识别, 隔离和处理的肺泡巨噬细胞从人和小鼠存在。这种方法是必要的研究这些重要的先天免疫细胞在各种实验设置。这里描述的方法可以很容易地被任何实验室采用, 是一种从支气管肺泡灌洗液或肺组织中获取肺泡巨噬细胞和维持它们的体外的简化方法。由于肺泡巨噬细胞主要发生在肺泡中的黏附体细胞, 所以这种方法的重点是在收获和鉴定之前逐出。肺是一种高度血管化的器官, 各种细胞类型的髓和淋巴源栖息, 相互作用, 并受肺部微环境的影响。通过使用这里描述的一组表面标记, 研究人员可以很容易和明确地区分肺泡巨噬细胞与其他白细胞, 并净化它们的下游应用。本发明的培养方法支持人和小鼠肺泡巨噬细胞用于体外生长, 并与细胞和分子研究相适应。

图 1显示了用于识别鼠标 AMs 的流细胞方法。这包括分析在区分 AMs 与其他肺居民或肺浸润吞噬必要的最低限度的表面标记。需要进行差异分析, 以积极识别细胞间质巨噬细胞、树突状体细胞、中性粒细胞、单核细胞以及在肺部发生的核型肺巨噬细胞。下面的曲面标记方案可用于在AMs为 CD45+的各个单元格之间方便地区分这?...

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

小鼠与人肺泡巨噬细胞的分离和体外培养

该通讯描述了从人类和小鼠模型中分离和培养肺泡巨噬细胞的方法, 以供实验之用。

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

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Immunology and Infection

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

23.6K 次观看.  St. Joseph's Hospital and Medical Center. 该通讯描述了从人类和小鼠模型中分离和培养肺泡巨噬细胞的方法, 以供实验之用。

视频: Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

生物工程

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

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

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

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

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

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

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

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. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

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

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.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

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

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

用于阿尔韦拉尔巨噬细胞和CD4和T细胞免疫造血和HIV储液评估的支气管性洗浴液和匹配血液的处理

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

从小鼠中分离和培养骨髓来源的巨噬细胞

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

小鼠窦房和房室结常驻心脏巨噬细胞的分离和培养

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...