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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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, 和低量的炎细胞因子, 如 IL-1223。一个信号来自于通过 Fc 伽玛受体作用的抗体, 第二个信号是炎刺激, 如 LPS 或 IFNγ24,25。血清, 免疫复合物, 抗 TNFα抗体, 都被发现诱导高 IL-10-produing 抗炎活化状态的巨噬细胞8,23,25,26。我们的小组以前发表过, 小鼠骨髓衍生的巨噬细胞和腹腔巨噬细胞刺激与丙种球蛋白也产生大量的 IL-10 和很少到没有炎 IL-12/23p40 响应 LPS8。我们还发现, 其他炎细胞因子, TNF 和 IL-6 也减少了免疫球蛋白在骨髓衍生巨噬细胞8。本文所描述的方法可用于检测小鼠 BMDMs 和腹腔巨噬细胞的其他抗体,体外和体内。
在小鼠骨髓和腹腔巨噬细胞的抗体治疗中, 有许多关键的步骤。保持无菌对每个协议都很重要。如果巨噬细胞的培养物被微生物从空气、毛皮或粪便中污染, 巨噬细胞就会通过产生炎细胞因子来反应这些刺激。在生物安全柜中进行所有实验, 用乙醇喷洒老鼠, 确保腹膜冲洗时肠道的完整性是必不可少的。抗体还必须保持不育, 并按照制造商的指示储存。它不能被使用, 如果它是污染, 过期, 或混浊。混浊会降低药物的有效性, 如果药物没有储存在适当的温度或贮存时间过长, 就会发生。丙种球蛋白可以储存在4° c, 只有在到期日达到之前使用, 因为 IL-10 生产的水平可能受到影响。或者, 我们已经看到, 它可以在-20 ° c 冻结, 对反应没有影响。不同的大量的免疫球蛋白导致类似的实验结果, 而不同类型的丙种球蛋白品牌的准备, 如在图 1, 可以导致不同的反应在巨噬细胞。必需的控制, 如刺激细胞, 和单独的抗体刺激的细胞, 必须包括在每个实验, 以排除巨噬细胞或药物的污染。
可以对这些协议进行若干修改, 以自定义一个实验。在性状态, 它可能是困难的分离足够的腹腔巨噬细胞为大实验。腹腔注射乙酸 (TG) 可诱发腹膜巨噬细胞。免疫应答发生, 并在4天后, 诱发腹膜巨噬细胞可以收获27。被招募的巨噬细胞可能不太成熟, 也不像我们在这里所用的那样代表常驻细胞, 这是重要的考虑因素。tg 诱发巨噬细胞也可能有内化琼脂的 tg 汤, 这可能是一个并发症, 特别是如果做实验的吞噬27。不同基因背景的小鼠, C57BL/6 或 balb/c, 也可用于实验, 并可选择检测抗体对巨噬细胞应答的影响, 这些反应将在需要特定遗传背景的疾病模型中进行评估。此外, 转基因小鼠, 如Il10/-小鼠, 可用于评估特定蛋白质在药物作用机制中的作用, 正如我们在我们自己的工作8中所做的那样。不同的炎症刺激也可以用来评估抗体对巨噬细胞活化的影响。IFNγ, 和主机衍生的 toll 样受体 (TLR) 激动剂 (如透明质酸), 已被用来激活 IL-10-producing 巨噬细胞25,28。一个重要的考虑因素是抗体的剂量。该剂量应滴定, 以获得最佳剂量的文化和动物实验, 因为它将不同的抗体和供应商。所选择的剂量也应反映给人类的剂量, 并将不同的药物。在高剂量 (25-35 毫克/毫升或2克/千克体重) 中, 静脉注射丙种球蛋白是一种抗炎治疗, 它反映了这些协议中使用的滴定剂量29,30。
骨髓和腹腔巨噬细胞的使用有其局限性。虽然在巨噬细胞系的改善, 小鼠 BMDMs 仍然是人工衍生的细胞从造血前体。检测巨噬细胞的免疫应答在体外是了解抗体应答的重要步骤, 但体内实验也需要进行。注射刺激在体内其次是培养细胞的前体, 可以为将来的研究提供更多的信息, 以研究抗体药物是否也能激活一种疾病状态下的抗炎巨噬细胞。关于接受抗体生物制剂的人所发生的事情的结论不能从这些实验中得出。很少有研究已经发表, 以评估免疫球蛋白对人单核细胞衍生巨噬体的影响在试管。IL-6 产生的人单核细胞的数量减少在体外已经报告, 当免疫球蛋白是 co-stimulated 与 lps 相比刺激与 lps 单独12。在 THP-1 人细胞细胞 (PCT) 中, 免疫球蛋白降低 TNFα和 IL-6 的产生,31。
本文中的协议优于现有的方法。骨髓衍生的巨噬细胞和腹腔巨噬细胞是主要的干细胞, 直接从小鼠中分离出来, 而不是永生化的细胞系。小鼠巨噬细胞像 RAW264.7 细胞一样, 不产生炎细胞因子, IL-12, 以回应 LPS, 这是一个重要的细胞因子, 是由 IL-10 调节的反应产生的免疫球蛋白8,13。通过对灌洗液、腹膜细胞、特别是腹腔巨噬细胞的分析, 检测药物在体内与 LPS 的结合允许检查药物对局部腹膜环境的影响。这是一个短期的, 简单的模型, 可以提供重要的信息, 以证明检查的非特异性作用的抗体对巨噬细胞活化的长期和更昂贵的动物模型的疾病。它的优势优于内毒素血症模型的实验措施往往只老鼠生存或细胞因子在血清14。存活率和血清细胞因子是免疫应答的重要措施, 但它们并没有显示出巨噬细胞所能具有的特殊作用。从挑战实验中分离和培养腹膜巨噬细胞, 表明抗体疗法对巨噬细胞功能有直接和可测量的作用。
这些方法在生物疗法的测试和设计中有应用。近年来, 作为疗法的抗体的使用急剧增加。然而, 这些治疗可能对巨噬细胞有额外的未知作用, 可以识别抗体的 Fc 部分和改变其细胞因子的产生。抗 TNFα抗体, 昔, 已被证明诱导 IL-10 和抑制 T 细胞增殖通过其 Fc 部分, 并提出作为其作用机制之一7,15,26。使用遗传模型, 特定的蛋白质可以牵连到这些药物如何影响巨噬细胞活化的机制。IgG 抗体和抗体的准备可以改变, 以改变如何影响巨噬细胞的生物制剂。我们的数据表明, 同一种抗体药物 (丙种球蛋白) 的制备和/或贮存会影响其对巨噬细胞活化的影响。本文中的方法可以用来设计没有这些效果的疗法, 这可能是在癌症治疗期间维持监视的关键, 在那里抗炎巨噬细胞可能促进肿瘤的进展。这些技术也可用于设计和评价药物, 如果有必要, 这些效果也有这些作用。例如, 它可能是有益的减少巨噬细胞炎症反应和创建抗炎 IL-10-producing 巨噬细胞, 以加强治疗炎症性肠疾病或类风湿关节炎。

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。

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			<td height="21" style="height:21px;width:453px;">Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12440053</td>
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			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
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			<td style="width:408px;">555252</td>
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			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9101</td>
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			<td height="21" style="height:21px;width:453px;">anti-pp38 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9211</td>
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			<td height="21" style="height:21px;width:453px;">anti-GAPDH antibody</td>
			<td style="width:209px;">Fitzgerald industries</td>
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			<td height="21" style="height:21px;width:453px;">26 g needle</td>
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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 刺激单独 (图 1A) 和减少生产 IL-12/23p40 (图1 B)。图 1还演示不同的丙种球蛋白制剂的执行方式有所不同。虽然三不同制剂的丙种球蛋白可以显著减少 IL-12/23p40 (图 1B), Octagam (奥) 和 Gammagard 液体 (GL), 不显着增加 IL-10 生产响应 LPS (图 1A). 和 GL 能够显著减少 IL-12/23p40 的生产, 以应对 LPS, 但在较小程度上比广西。这些结果表明, 抗体影响骨髓衍生巨噬细胞活化, 并有不同的准备相同的药物, 可导致反应的变化。
BMDMs 可以用来测试的机械效应, 静脉注射免疫球蛋白, 由西方印迹。与 IC + LPS 激活的巨噬细胞增加磷酸化的丝裂原活化蛋白 (MAP) 激酶, p38 和 Erk1/2, 这是负责增加 IL-10 生产21。在图 2中的结果显示了一个典型的西方印迹, 用于检测刺激的巨噬细胞 IL-10 生产所需的 MAP 激酶激活, 或者被 lps、丙种球蛋白或丙种球蛋白 + lps 刺激。单独免疫球蛋白或丙种球蛋白 + lps 刺激增加活化的 MAP 激酶, Erk1/2, 与早期和长期的磷酸化相比, 单独看到的 lps。p38 的激活发生在早期的巨噬细胞刺激与免疫球蛋白 + lps 相比, 仅刺激与 lps 单独。这些结果表明, 方法, 如印迹, 可以用来显示细胞信号的作用, 抗体药物的 BMDMs。除了细胞因子的生成, MAP 激酶活化可用于显示抗炎巨噬细胞的活化发生。
成熟的, 组织驻留的巨噬细胞也可以从小鼠中分离出来, 以评估对免疫球蛋白的反应 5.0 x 105 -1.0 x 106腹腔巨噬细胞可以从一个健康的 C57BL/6 老鼠中分离出来。在图 3中, 与 PBS 注射的小鼠相比, 注射丙种球蛋白的小鼠腹腔巨噬细胞在没有刺激的情况下, 不会显著增加 IL-10 的产量. 当注射静脉注射小鼠的腹腔巨噬细胞与 LPS前体刺激时, 它们比注射 PBS 的小鼠产生6倍以上的 IL-10。然而, 当与 LPS前体刺激时, 它们不会产生可检测的 IL-12/23p40 量。这些结果表明, 抗体的作用可以测试的巨噬细胞注射药物在体内和培养细胞前体内的方法。
巨噬细胞对抗体和炎症刺激的反应可以在体内进行评估。细胞因子的产生可以在灌洗液中进行评估, 而在清从前体内刺激腹膜巨噬细胞。图 4显示了在灌洗液 (图 4A)、腹膜细胞 (图4B) 和腹膜巨噬瘤 (图 4C) 中, 小鼠的抗炎细胞因子 IL-10 的增加与 PBS + lps 相比较, 有免疫球蛋白 + lps 的挑战。炎细胞因子亚基 IL-12/23p40 在培养的腹腔巨噬细胞中明显减少, 但在灌洗液中不存在。这种方法允许检查抗体药物对炎症反应的影响在体内以及ex 体内。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56689/56689fig1.jpg"> 
图 1: 巨噬细胞 IL-10 和 IL-12/23p40 的生产反应刺激与不同品牌的丙种球蛋白和 LPS.C57BL/6 MCSF 骨髓源性巨噬细胞要么刺激 (C), 要么刺激与 LPS (10 ng/毫升), 丙种球蛋白 (30 毫克/毫升), 或免疫球蛋白 + LPS。对三种不同牌子的丙种球蛋白进行了测试: Gammunex (广西)、Gammagard 液 (GL) 和 Octagam。在刺激后收集24小时巨噬细胞清, 经离心澄清。执行了 IL-10 (A) 和 IL-12/23p40 (B) 的 ELISAs。数据是从 n = 3 独立实验的巨噬细胞, 每实验1个单独的小鼠, 与 ELISAs 检测重复。数据是指± *p和 #60; 0.05, ** p和 #60; 0.001, ***p和 #60; 0.0001 为 lps 刺激与丙刺激的比较。对方差 (方差分析) 与 Tukey 的后测试进行了多次比较, 用于统计分析。<a href="//ecsource.jove.com/files/ftp_upload/56689/56689fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56689/56689fig2.jpg"> 
图 2: 免疫球蛋白活化巨噬细胞中 MAP 激酶活化的印迹分析.MCSF 衍生的骨髓巨噬细胞是刺激或刺激与 lps (10 ng/毫升), 广西静脉免疫球蛋白 (30 毫克/毫升), 或免疫球蛋白 + lps;为0、10、40或120分钟. 全细胞裂解 (1.0 x 106巨噬细胞/泳道) 受十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS 页) 和磷特异性抗体 p38 和胞外的印迹信号调节激酶 (Erk1/2), 以及甘油3磷酸脱氢酶 (GAPDH), 作为一个加载控制。结果显示为 n = 3 独立实验, 每实验1单个小鼠的细胞。pp38 和 pErk1/2 蛋白水平的密度, 正常化到 GAPDH, 平均从3独立的实验显示在每个波段下面。此图已从 Kozicky et al8中修改。<a href="//ecsource.jove.com/files/ftp_upload/56689/56689fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56689/56689fig3.jpg"> 
图 3: IL-10在体内使用免疫球蛋白的小鼠的巨噬细胞生产 ".8周大的 C57BL/6 小鼠被给予无菌 PBS (注射控制) 或共免疫球蛋白 (2.5 克/千克) 腹腔。1小时后, 对小鼠进行了安乐死和腹膜 lavages。腹腔巨噬细胞采用黏附选择分离。巨噬细胞要么是刺激 (C), 要么被 LPS 刺激 (10 ng/毫升)。24小时后, IL-10 ELISAs 细胞清的收获和澄清。数据是从 n = 5 个别小鼠每组执行在3独立的实验, 与 ELISAs 的化验重复。数据是指± * P和 #60; 0.01 和 NS = 不显著。统计分析是使用 Sidak 的前后进行多重比较的两种方式方差。此图已从 Kozicky et al8中修改。<a href="//ecsource.jove.com/files/ftp_upload/56689/56689fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56689/56689fig4.jpg"> 
图 4: IL-10 和 IL-12/23p40 小鼠在体内的免疫球蛋白 + LPS 的生产,.8周老 C57BL/6 小鼠被注射腹腔与 PBS + LPS (0.2 µg/克体重), 作为一个控制;(2.5 克/千克体重) + LPS (0.2 µg/克体重)。1小时后, 对小鼠进行了安乐死和腹膜 lavages。(A) 澄清了灌洗液, (B) 澄清了在 1 h 巨噬细胞黏附步骤期间腹膜细胞的条件清, (C) 澄清了 24 h 腹膜巨噬细胞 (Mφ) 清IL-10 和 IL-12/23p40 的 ELISA。数据是指± SD 为 n = 5 小鼠在3独立实验中, 与 ELISAs 的化验重复。*p和 #60; 0.05, **p和 #60; 0.01, ***p和 #60; 0.001, 和 NS = 不显著。用 PBS + lps 注射的小鼠与使用学生的t测试的静脉注射免疫球蛋白 + lps 的小鼠进行比较。此图已从 Kozicky et al8中修改。<a href="//ecsource.jove.com/files/ftp_upload/56689/56689fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.1K 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

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

科研

免疫与感染

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

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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