力是不列颠哥伦比亚大学4年奖学金 (4YF) 研究生李兆基奖获得者。L.M.S. 是加拿大胃肠病学/克罗恩病和结肠炎加拿大协会/研究院新调查员薪资奖的接受者, 是迈克尔. 史密斯健康研究基金会的生物医学学者。这项工作得到加拿大血液服务项目赠款的支持, 与加拿大卫生研究所合作 (赠款 CIHR2016-LS)。

这里描述的所有方法都得到了不列颠哥伦比亚大学机构动物保育和使用委员会 (IACUC) 的批准。
1. 骨髓巨噬细胞的抗体衍生与活化
<ol>
	<li>使用 CO2窒息执行安乐死。<ol>
		<li>将鼠标置于感应腔内。安乐小鼠与5% 异氟醚麻醉, 60-九十年代, 直到静止和呼吸是深和缓慢。</li>
		<li>关闭异氟醚麻醉并管理 CO2的 6-8 升/分, 直到鼠标停止呼吸。将鼠标放在 CO2上, 至少再使用5分钟. 验证不再有心跳或呼吸。执行颈椎脱位以确保死亡。 注: 8-12 周大鼠提供了巨噬细胞的最高产量。</li>
	</ol>
	</li>
	<li>用70% 乙醇喷洒老鼠腿, 用胳膊和腿把它们钉在泡沫板的仰卧位上。用剪刀和钳子来保持皮肤, 做一个浅切 (0.2 厘米), 以去除皮肤和皮毛从表面的每一条后腿。 注意: 在无菌罩中执行此过程和所有组织培养操作。</li>
	<li>修剪肌肉使胫骨和股骨可见。去除胫骨和股骨, 通过切断髋部和踝关节下方的骨骼和肌肉。尽可能多地修剪肌肉。</li>
	<li>喷雾骨与70% 乙醇和等待1分钟, 它蒸发, 然后放置在6井板骨冲洗介质 (Iscove 氏改良 Dulbecco 的培养基 (IMDM), 10% 胎牛血清 (FBS))。</li>
	<li>修剪骨骼的两端, 从踝关节和股骨顶端切下0.1 厘米的骨, 使骨腔暴露。确保骨髓是可见的, 26 口径的针可以放在骨腔内。</li>
	<li>切开骨骼和肌肉, 将胫骨和股骨从膝关节中分离出来。将一个26口径的针头插入一个10毫升的注射器进入腔内。将骨髓冲洗成50毫升的锥形管, 5 毫升的骨冲洗培养基。冲洗两个胫骨和两个股骨, 从一个鼠标, 到每50毫升管。</li>
	<li>吸管向上和向下几次或漩涡的骨髓在缓慢的速度, 轻轻地分散团簇。骨髓的细绳应该被分解成小 (少于0.5 毫米) 的骨髓斑点。用骨冲洗介质将锥形管装入50毫升。吸管的内容的锥形管成75厘米2组织培养处理烧瓶, 并孵化在37° c, 5% CO2为 1 h。 注: 成熟的巨噬细胞和间充质细胞将成为附着的烧瓶和被丢弃, 而所需的造血祖保持在悬浮。</li>
	<li>将造血祖细胞移入50毫升锥形管中。在室温下离心 300 x g 的管, 5 分钟. 在5毫升的巨噬细胞集落刺激因子 (MCSF) 培养基中丢弃上清和重颗粒 (IMDM, 10% FBS, 5 ng/毫升 MCSF, 100 U/毫升青霉素/链霉素, 和150μ mmonothioglycerol (MTG))。</li>
	<li>使用例18对单元格进行计数。将培养基添加到重细胞中, 浓度为 0.5 x 106细胞/毫升, 1.5 x 107细胞/烧瓶在30毫升培养基中, 吸管上下轻轻地向上和向下。吸管30毫升的悬浮到一个新的75厘米2组织培养处理烧瓶。</li>
	<li>在4天和7天的初始培养后, 在步骤 1.9, 验证细胞的粘附和轻微分支使用倒置相衬明亮的现场显微镜。丢弃包含换药单元格的介质。一次用 IMDM 冲洗细胞, 加入30毫升的 MCSF 培养基。</li>
	<li>当细胞是成熟的10天 (和 #62; 95% 阳性 F4/80 和 Mac-1), 再次验证细胞是粘附和轻微。</li>
	<li>对细胞进行刺激, 从烧瓶中取出培养基, 加入8毫升的酶释放, EDTA-based 细胞离解缓冲 (材料表)。孵育细胞为5分钟在37° c, 5% CO2。</li>
	<li>轻轻地刮起细胞, 用少量的压力, 用无菌的细胞刮板。在显微镜下检查细胞与烧瓶表面分离。吸管分离细胞成一个50毫升锥形管。用5毫升的 IMDM 冲洗烧瓶3次, 并将冲洗液与所收获的细胞池。在室温下离心 300 x g 的细胞5分钟。</li>
	<li>重3毫升的 MCSF 培养基每50毫升圆锥管的细胞颗粒。使用例18计算可行单元格的个数。重细胞的浓度为 1 x 106细胞/毫升和板100μ l 细胞悬浮 (1 x 105细胞/井) 每井的 96-良好组织培养处理, 平底板。 注: 从一只老鼠的骨骼将产生足够的细胞为100口井。如果需要, 1 毫升的细胞可以镀在一个6井板 (组织培养处理, 平坦的底部)。较大数量的单元格 (1 x 106单元格) 可能对西部印迹分析有用。</li>
	<li>一旦贴附, 刺激重复或三份井每一个与 10 ng/毫升的 LPS 在 IMDM, 30 毫克/毫升的丙种球蛋白, 或丙种球蛋白 + LPS。剂量的 LPS 或丙种球蛋白可以滴定, 以优化反应。留下重复或三份井作为刺激控制。孵育他们为24小时 (37 ° c, 5% CO2)。 注: 再镀后的细胞应在1小时内坚持组织培养井。</li>
	<li>收集细胞清从每个井到单独的1.7 毫升离心管和去除任何颗粒物质通过纺纱在 1万 x g 5 分钟。</li>
	<li>取出细胞上清液, 避免扰乱颗粒。将澄清的细胞上清液放置在无菌离心管中。 注意: 细胞清可以检测细胞因子, IL-10, 细胞因子亚基, IL-12/23p40, 酶联免疫吸附试验 (ELISA) 立即, 或储存在-80 ° c 长期。遵循 ELISA 协议从商业可利用的成套工具 (材料目录)。其他炎细胞因子可以检测, 如 IL-6 和 TNF, 因为他们也减少了 co-treatment 与免疫球蛋白 + lps 刺激相比, 单独刺激与 lps。在刺激之后, 黏附细胞可以为技术准备, 例如西部印迹或定量聚合酶链式反应 (Q PCR) 19,20。</li>
</ol>
2. 具有挑战性的小鼠在体内与静脉注射和收获腹腔巨噬细胞
<ol>
	<li>称量每只老鼠。</li></ol>计算每只老鼠所需的2.5 克/千克体重的最终剂量时, 需要的静脉注射丙种球蛋白的体积 (100 毫克/毫升的浓度)。 注: 例如, 对于20克鼠标, 需要500µL 的丙种球蛋白。
	<li>将所需量的丙种球蛋白放入不育的1毫升注射器中, 并装有无菌26口径针头。对于不接受丙种球蛋白的控制小鼠, 管理一个等效剂量容积的无菌磷酸缓冲盐水 (PBS), pH 值7.4 而不是静脉注射。</li>
	<li>用你的拇指和食指抓住它的皮肤, 用你剩余的手指握住它的尾巴和后腿。将身体向地面倾斜。</li>
	<li>将针放在 30-40 °角相对于鼠标的腹部, 在右下象限, 向上倾斜, 并插入1.5 厘米的针头。注射免疫球蛋白或 PBS 腹腔。等1小时</li>
	<li>安乐5% 异氟醚麻醉的小鼠, 其次为 6-8 升/分 CO2窒息 (见步骤 1.1.1-1.1.2), 或根据机构的道德准则。</li>
	<li>将鼠标钉在泡沫板上, 用四肢展开。用70% 乙醇喷洒零件。 注意: 在无菌罩中执行程序。</li>
	<li>用剪刀沿着小鼠的中线在皮肤 (0.2 厘米) 上做一个浅切口, 以避免切割腹膜腔。用钳子把腹部的皮肤从老鼠的腹部拉下来, 使腹膜壁的衬里可见。</li>
	<li>用5毫升无菌 PBS (pH 7.4) 填充5毫升注射器。将25或27口径的针插入腔的顶部, 从左侧或右侧向鼠标的中心, 用针的斜面向上。 注意: 将针小心地放在鼠标上, 这样就不会将 PBS 注入器官, 而是放入腹膜间隙。</li>
	<li>将液体推入腹膜腔。将针头从腔中拔出, 并按摩十年代的腹膜, 以取出任何细胞。在腔顶部插入针, 避免器官。收集并放置在冰上的15毫升锥形管回收灌洗液。 注: 每5毫升的液体注入, 大约3.75 毫升将恢复。</li>
	<li>重复注射和恢复 (步骤 2.8-2.9) 3 次 (总注射量20毫升), 以恢复15毫升的灌洗液的总和。从每只老鼠收集灌洗成一个单独的15毫升锥形管。 注: 在最后一次注射后, 在液体积聚的地方, 从鼠标的最左、右两侧提取液体可能更容易。在这个位置上, 器官会对针头产生较少的干扰。</li>
	<li>在4° c 下, 在 300 x g 处旋转细胞5分钟。重的细胞在500μ l 的电镀介质 (IMDM, 10% FBS, 和100的 U/毫升青霉素/链霉素) 每只老鼠刷新。使用例18计算可行的巨噬细胞。 注意: 巨噬细胞会比其他的腹膜細胞略大。典型的收益率是 5 x 105 -1 x 106巨噬细胞/鼠标使用野生类型的 C57BL/6 鼠标。 可选: 如果细胞颗粒中存在大量的红细胞, 则根据制造商的说明, 使用1x 红细胞裂解缓冲液, 执行溶解步骤去除这些细胞。</li>
	<li>重细胞在电镀介质中在 1 x 106巨噬体/mL 和板100μ l 每井在96井平底组织培养处理的板材。 注意: 如果使用一份三口井或滴定兴奋剂, 可能需要将多个鼠标的细胞汇集在一起进行实验。例如, 如果一个鼠标有 5 x 105巨噬细胞, 将需要500μ l 的电镀介质。将有足够的细胞为5口井, 或1试验重复, 一 LPS 剂量。</li>
	<li>孵育细胞为1小时在37° c, 5% CO2 , 以使噬细胞成为附着。黏附细胞是腹腔巨噬细胞膜。移除单元格清和换药单元格。冲洗两次井用200μ l IMDM (5ml 到37° c), 等待十年代和慢慢地倾斜板材。更换电镀介质。孵育细胞在37° c, 5% CO2在刺激之前30分钟。</li>
	<li>刺激巨噬细胞与 10 ng/毫升的 LPS 在 IMDM, 或离开刺激, 作为一个控制。孵育24小时, 收集和澄清细胞清 (见步骤 1.16-1.17) IL-10 细胞因子分析的 ELISA。遵循 ELISA 协议从商业可利用的成套工具 (材料目录)。 注: 在刺激后, 贴壁细胞可以为诸如免疫印迹或 PCR 等技术而准备。</li>

3. 具有挑战性的小鼠在体内与免疫球蛋白 + LPS 和收获腹膜巨噬细胞
<ol>
	<li>称量每只老鼠。计算所需的丙种球蛋白 (100 毫克/毫升的浓度) 的体积, 以提供最终剂量的2.5 克/千克体重。计算所需的 LPS (100 微克/毫升的库存浓度在 IMDM), 以提供最后剂量的0.2 微克/克体重。 注: 例如, 对于20克小鼠, 500 µL 的丙种球蛋白库存溶液和40μ l 的100微克/毫升 LPS 溶液的需要。</li>
	<li>将所需量的丙种球蛋白和 LPS 注射到不育的1毫升注射器中, 并装有无菌26口径针头。对于不接受丙种球蛋白的控制小鼠, 用无菌 PBS pH 值7.4 代替等量的免疫球蛋白。</li>
	<li>用你的拇指和食指抓住它的皮肤, 用你剩余的手指握住它的尾巴和后腿。将身体向地面倾斜。</li>
	<li>将针放在 30-40 °角相对于鼠标的腹部, 在右下象限, 向上倾斜, 并插入1.5 厘米. 注射丙型球蛋白 + lps, 或 PBS + lps, 腹腔在步骤2.4。</li>
	<li>1h 后, 通过执行步骤 2.5-2.10, 收获腹腔巨噬细胞。</li>
	<li>在4° c 下, 在 300 x g 处旋转细胞5分钟。去除1毫升的回收灌洗液, 并用于 IL-10 和 IL-12/23p40 分析的 ELISA 根据制造商的说明或冻结在-80 ° c 的未来分析。 注: 为确保对灌洗液细胞因子分析的治疗进行准确比较, 5 毫升 flushs 腹膜腔4次, 最后一卷15毫升。并不是所有的液体都会从每个冲洗液中恢复。</li>
	<li>在步骤2.11 中, 重和计数可行的巨噬细胞。 注意: 巨噬细胞会比其他的腹膜細胞略大。</li></ol>典型的收益率是 5 x 105 -1 x 106巨噬细胞/鼠标使用野生类型的 C57BL/6 鼠标。 可选: 如果在细胞颗粒中存在大量的红细胞, 按照制造商的说明执行裂解步骤, 如步骤2.11 所示。
	<li>如步骤2.12 所示, 在电镀介质中 re-suspend 细胞。 注意: 可能需要从多个鼠标池中的单元格进行实验。例如, 如果一个鼠标有 5 x 105巨噬细胞, 将需要500μ l 的电镀介质。</li>
	<li>在 2.13, 孵育细胞为 1 h 在37° c 5% CO2允许巨噬细胞膜成为黏附。</li>
	<li>收集和澄清条件培养基, 每步骤 1.14-1.15, 从所有腹膜细胞。 注: 样品可立即使用或冷冻保存在-80 ° c, 用于细胞因子分析。</li>
	<li>执行步骤 2.11, 但孵育24小时刺激细胞。收集和澄清细胞清, 根据步骤 1.16-1.17, 细胞因子分析的 ELISA。 注: 细胞可以为诸如西方印迹或 PCR 等技术而准备, 如步骤 1.16-1.17 和2.14。</li>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+uses+of+intravenous+immunoglobulin.">Clinical uses of intravenous immunoglobulin.</a> Clin Exp Immunol. 142 (1), 1-11 (2005).</li><li>Murakami, 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=Intravenous+immunoglobulin+preparation+prevents+the+production+of+pro-inflammatory+cytokines+by+modulating+NF&#954;B+and+MAPKs+pathways+in+the+human+monocytic+THP-1+cells+stimulated+with+procalcitonin.">Intravenous immunoglobulin preparation prevents the production of pro-inflammatory cytokines by modulating NF&#954;B and MAPKs pathways in the human monocytic THP-1 cells stimulated with procalcitonin.</a> Inflamm Res. 63 (9), 711-718 (2014).</li></ol>

作者没有利益冲突透露。

巨噬细胞活化状态在组织稳态和疾病中起重要作用22。巨噬细胞可以有不同的和重叠的活化状态, 根据他们的微环境的提示3。它们在炎症反应的各个阶段都有不同的作用: 防御病原体, 伤口愈合和组织恢复, 也有明显的抗炎活化状态, 对于关闭炎症反应很重要2.抗炎巨噬细胞激活需要两个外部刺激巨噬细胞产生大量的抗炎细胞因子, IL-10, 和低量的?...

巨噬细胞是先天免疫的, 在免疫应答中扮演多重角色, 感染或受伤。巨噬细胞负责启动免疫应答感染或组织损伤, 停止炎症反应, 并促进愈合响应1。三最佳研究的巨噬细胞活化状态的例子是: 1) 巨噬细胞治疗干扰素伽玛 (IFNγ) 和细菌脂多糖 (lps), 指定 M (IFNγ + lps), 导致炎症反应;2) 用白细胞介素 4 (IL-4)、M (IL-4) 刺激的巨噬细胞, 与愈合应答有关;3) 巨噬细胞刺激与免疫复合体 (ic) 和 lps, M (ic + lps), 有能力关闭炎症反应2,3。m (IC + LPS) 是不同的 m (IL-4) 伤口愈合巨噬细胞, 并没有表达的酶酸 (Arg-1) 或 FIZZ14。这些抗炎巨噬细胞的最佳标记是其细胞因子的生成5。巨噬细胞在维持健康方面有多种作用, 但也会导致炎症性疾病和癌症3。因此, 巨噬细胞是治疗各种疾病的关键治疗靶。重要的是要研究抗体对其活化状态的影响, 发展 macrophage-based 治疗疾病。
本文的重点是利用小鼠骨髓源性巨噬细胞 (BMDMs) 和腹腔巨噬细胞检测抗体药物对炎症反应的影响体外和体内。最近, 有多项研究显示抗体对巨噬细胞激活的影响6,7,8。巨噬细胞 co-activated 与免疫配合物, 是抗体与抗原复合, 和 LPS, 通常炎症刺激, 产生非常高水平的抗炎细胞因子, IL-10, 和非常低的水平的炎细胞因子,白细胞介素 12 (IL-12)9。此外, 昔, 一个抗 TNFα单克隆抗体, 已发现工作, 部分, 通过诱导抗炎巨噬细胞通过其片段结晶 (Fc) 区域7。我们已经报告, 丙种球蛋白 + lps 诱导抗炎巨噬细胞活化, 类似于 M (IC + lps), 其中 co-stimulated 巨噬细胞产生大量的 IL-10 和低量的炎细胞因子亚基白细胞介素12或 23 p40 (IL-12/23p40), 白细胞介素-6 (IL-6), 和 TNF8。丙种球蛋白是一种由多克隆抗体组成的药物, 主要是 IgG, 从1000多个捐献者的血液中汇集而来10。它被用于治疗各种各样的免疫疾病, 例如特发性血小板减少紫癜和慢性脱髓鞘神经病, 但它的作用机制不完全地被了解11。抗体基础药物对巨噬细胞活化的影响可以用本文所描述的方法进行评估。
特定生物制剂对巨噬细胞活化的影响可在 BMDMs 和腹腔巨噬细胞中检测。使用这些巨噬细胞来源, 可对原体细胞进行评估。对原代细胞抗体的初步检测比其他耗时和昂贵的疾病模型需要更少的时间和金钱投资。通过将药物注入健康的鼠标在体内, 并隔离这些细胞并分析它们的体外, 可以确定是否有必要进行研究以评估生物制剂是否会影响巨噬细胞在疾病模型中的活化作用. 
很少有研究测试生物疗法对巨噬细胞活化的影响在体外和在体内直接, 我们的技术提供了比替代技术的优势。目前的技术包括测试生物药物对混合细胞数量的影响在体外, 如昔在混合淋巴细胞反应 (MLR) 或静脉注射丙种球蛋白对人巨噬细胞外周血单个核的影响不能归因于特定的单元格类型7,12。使用 BMDMs 和腹腔巨噬细胞是有利的, 在使用单元格线, 如 RAW264.7 细胞, 不产生炎细胞因子, IL-12, 以响应 LPS8,13。检测抗体药物对巨噬细胞反应的影响ex 体内具有优势, 因为细胞因子反应可以直接归因于巨噬细胞, 而不是通过测量血清细胞因子水平来推断巨噬细胞的反应,14.BMDMs 和腹腔巨噬细胞可以获得和分离的基因修饰小鼠, 以确定特定的作用, 一个蛋白质的抗炎巨噬细胞活化。例如, 我们使用了Il10缺陷的(--) BMDMs 来证明静脉注射丙种球蛋白诱导的炎细胞因子的减少是部分依赖于 IL-108。药物的作用机制可以用西方印迹来进行研究, 在那里可以确定特定蛋白质和信号事件的角色。定量聚合酶链反应 (qPCR) 可以在 BMDMs 或腹腔巨噬细胞上进行, 以显示抗体活化引起的基因表达模式。小鼠疾病模型可以提供关于抗体生物疗法在炎症性肠病、类风湿关节炎和癌症等疾病模型中的潜在功效的信息15,16,17. 然而, 这里描述的技术将提供关于这些生物制剂的作用机制的信息通过确定他们是否导致抗炎巨噬细胞活动。

<table><tbody><tr><td>Iscove&amp;rsquo;s modified Dulbecco&amp;rsquo;s medium (IMDM)</td><td>Life technologies</td><td>12440053</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life technologies</td><td>12483-020</td><td></td></tr><tr><td>Recombinant murine macrophage colony stimulating factor (MCSF)</td><td>Stemcell technologies</td><td>78059</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Life technologies</td><td>15140148</td><td></td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma</td><td>88640</td><td></td></tr><tr><td>1X red blood cell lysis buffer</td><td>eBioscience</td><td></td><td></td></tr><tr><td>Cell dissociation buffer</td><td>Life technologies</td><td>13150016</td><td>Enzyme-Free, Hanks&#039;s-based, EDTA</td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Sigma aldrich</td><td>L 4516</td><td>From E. coli 0127:B8</td></tr><tr><td>IVIg (Gammunex)</td><td>Grifols</td><td>Received from BC Children&#039;s Hospital, Transfusion Medicine</td><td></td></tr><tr><td>IVIg (Gammagard liquid)</td><td>Baxter Healthcare Corporation</td><td>Received from BC Children&#039;s Hospital, Transfusion Medicine</td><td></td></tr><tr><td>IVIg (Octagam)</td><td>Octapharma</td><td>Received from BC Children&#039;s Hospital, Transfusion Medicine</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) (sterile), pH 7.4</td><td>Life technologies</td><td>10010023</td><td></td></tr><tr><td>mouse IL-10 ELISA</td><td>BD biosciences</td><td>555252</td><td></td></tr><tr><td>mouse IL-12/23p40 ELISA</td><td>BD biosciences</td><td>555165</td><td></td></tr><tr><td>anti-Erk1/2 antibody</td><td>Cell signalling technology</td><td>9101</td><td></td></tr><tr><td>anti-pp38 antibody</td><td>Cell signalling technology</td><td>9211</td><td></td></tr><tr><td>anti-GAPDH antibody</td><td>Fitzgerald industries</td><td>10R-G109A</td><td></td></tr><tr><td>26 g needle</td><td>BD biosciences</td><td>305110</td><td></td></tr><tr><td>1 mL syringe</td><td>BD biosciences</td><td>309659</td><td></td></tr><tr><td>10 mL syringe</td><td>BD biosciences</td><td>309604</td><td></td></tr><tr><td>15 mL conical tube</td><td>BD biosciences</td><td>352096</td><td></td></tr><tr><td>50 mL conical tube</td><td>BD biosciences</td><td>352070</td><td></td></tr><tr><td>microcentrifuge tube (1.7 mL)</td><td>Diamed</td><td>SPE155-N</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2 &lt;/sup&gt;tissue culture treated flask</td><td>BD biosciences</td><td>353136</td><td></td></tr><tr><td>Cell scraper</td><td>BD biosciences</td><td>353085</td><td></td></tr><tr><td>Forcepts</td><td>VWR</td><td>82027-386</td><td>fine tip, dissecting&amp;nbsp;</td></tr><tr><td>Scissors</td><td>VWR</td><td>82027-582</td><td>Delicate, 4 &lt;sup&gt;1&lt;/sup&gt;/&lt;sub&gt;2&lt;/sub&gt;&quot;</td></tr><tr><td>Brightfield microscope</td><td>Motic</td><td>AE31</td><td>Inverted phase contrast</td></tr><tr><td>Scale&amp;nbsp;</td><td>Mettler&amp;nbsp;</td><td>PE 3000</td><td></td></tr></tbody></table>

assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation

巨噬细胞是吞噬先天免疫细胞, 它启动免疫反应的病原体和贡献愈合和组织恢复。巨噬细胞在关闭炎症反应方面同样重要。我们已经表明, 静脉注射免疫球蛋白 (丙种球蛋白) 刺激的巨噬细胞可以产生大量的抗炎细胞因子, 白细胞介素 10 (IL-10), 和低水平的炎细胞因子响应细菌多糖 (LPS)。丙种球蛋白是一种多抗体, 主要是免疫球蛋白 Gs (IgGs), 从血浆中汇集超过1000献血者。它是用来补充免疫缺陷患者的抗体或抑制免疫反应的患者自身免疫或炎症条件。昔, 一个治疗性抗肿瘤坏死因子α (TNFα) 抗体, 也被证明激活巨噬细胞产生 IL-10 反应炎症刺激。免疫球蛋白和其他抗体生物制剂可以通过测试来确定它们对巨噬细胞活化的影响。本文介绍了在体外抗体激活的小鼠骨髓巨噬细胞的衍生、刺激和评价方法, 以及抗体激活的小鼠腹腔巨噬细胞 (体内。最后, 我们展示了利用西方印迹来确定特定细胞信号通路对抗炎巨噬细胞活动的贡献。这些协议可以与基因修饰的小鼠一起使用, 以确定特定的蛋白质对抗炎巨噬细胞活化的影响。这些技术也可以用来评估特定的生物制品是否可以通过改变巨噬细胞的 IL-10-producing 抗炎活化状态, 减少炎症反应在体内。这可以提供关于巨噬细胞活化在动物疾病模型中的作用的信息, 并提供对人类潜在的新的行动机制的洞察力。反之, 这可能告诫不要使用特定的抗体生物制剂来治疗传染性疾病, 特别是如果巨噬细胞在宿主防御感染中起重要作用。

小鼠骨髓源性巨噬细胞可以从骨髓物的造血细胞前体中培养。骨髓物池从股骨和胫骨的一个 C57BL/6 鼠通常产生 107骨髓衍生巨噬细胞, 使他们成为一个方便的来源的巨噬细胞的实验。BMDMs 可用于测试抗体的药物反应时, 挑战与炎症刺激体外。图 1显示了丙种球蛋白品牌, Gammunex (广西), + lps 增加的抗炎细胞因子的生产, IL-10, 7 倍与 lps 刺激单...

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Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

抗体药物对炎症性疾病进行了革命性的治疗。除了对特定靶点产生直接影响外, 抗体还能激活巨噬细胞成为抗炎药。该协议描述了如何抗炎巨噬细胞活化可以评估在体外,使用小鼠骨髓巨噬细胞, 和在体内,使用腹腔巨噬细胞。

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

assessment-antibody-based-drugs-effects-on-murine-bone-marrow

Research

JoVE Journal

Immunology and Infection

8.2K 次观看.  University of British Columbia. 抗体药物对炎症性疾病进行了革命性的治疗。除了对特定靶点产生直接影响外, 抗体还能激活巨噬细胞成为抗炎药。该协议描述了如何抗炎巨噬细胞活化可以评估在体外,使用小鼠骨髓巨噬细胞, 和在体内,使用腹腔巨噬细胞。

视频: Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

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 ...

科研

生物工程

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

免疫与感染

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

抗结核抗体对骨细胞形成和骨细胞前体增殖的影响

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

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 ...

新生儿骨髓来源巨噬细胞的吞噬试验评价 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

作者聚焦：开发一种从新生小鼠中分离骨髓来源的巨噬细胞的可重复方法，以促进早期免疫反应

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

使用单核细胞-巨噬细胞测定预测红细胞抗体的意义

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

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此视频中的章节

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

在小鼠体内的巨噬细胞的枯竭和重建

骨髓衍生的巨噬细胞产

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

抗结核抗体对骨细胞形成和骨细胞前体增殖的影响

从小鼠中分离和培养骨髓来源的巨噬细胞

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

使用单核细胞-巨噬细胞测定预测红细胞抗体的意义