Name: bronchoalveolar lavage of murine lungs to analyze inflammatory cell infiltration
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左心室肥厚是在根特大学生物医学系的分子生物学研究助理。 ERJ由UniVacFlu支持，授权号607690. KR由EC-FP7项目FLUNIVAC支持。

本研究中描述的所有动物实验都是根据国家（比利时第14/08/1986号和第22/12/2003号比利时皇家法令06/04/2010）和欧洲立法（欧盟指令2010/63 / EU和86 / 609 / EEC）。所有对小鼠和所有动物方案的实验均经根特大学伦理委员会批准（许可证编号LA1400091和EC2016-027）。 准备<ol><li>灌洗液<ol><li>用100μM乙二胺四乙酸（EDTA）制备平衡的盐溶液。 注意:为了测量BAL液中的蛋白质水平，建议添加蛋白酶抑制剂以防止BAL液中的蛋白酶活性。 </li></ol></li><li>导管<ol><li>通过将23 G针插入透明塑料聚乙烯21 G管（内径:0.58 mm，外径:0.965 mm，长度:0.5 cm）制作导管。预制导管也可以使用。 </li></ol></li><li>麻醉外墙外保温系统<ol><li>制备末端麻醉剂，优选引起呼吸停止的终末麻醉剂（ 例如巴比妥酸盐，如磷酸缓冲盐水（PBS）中的戊巴比妥钠（&gt; 100 mg / kg）溶液）。 注意:建议使用注射麻醉而不是吸入麻醉，因为吸入麻醉可能会影响BAL液体含量。 CO 2例如对血液的pH具有影响，并因此影响不同化合物12的再分配。 </li></ol></li><li>氯化铵 - 钾（ACK）红细胞裂解缓冲液<ol><li>通过将8.29g NH 4 Cl和1g KHCO 3溶解在1L H 2 O中用100μMEDTA来制备ACK裂解缓冲液;红细胞裂解缓冲液也可以从外部来源购买。 </li></ol></li></ol> 2.进行支气管肺泡灌洗（BAL） <ol><li>将导管引入气管<ol><li>通过使用26 G针腹腔注射致死剂量的短效巴比妥酸盐麻醉来安乐死小鼠。要确认正确的致死性麻醉，用镊子夹住鼠标的后爪以检查脚反射。 </li><li>将动物放在手术板的背上，并固定鼠标。 </li><li>在颈部喷洒70％乙醇消毒。使用手术刀在气管附近的颈部皮肤切开。 </li><li>打开皮肤露出唾液腺。用钳子分开唾液腺以暴露胸骨肌。使用钳子切开气管周围的肌肉以暴露气管。 </li><li>使用钳子将棉线放在气管下。 </li><li>用26 G针，小心地穿刺两个软骨环之间的暴露气管中部。注意不要再损伤气管。 </li><li>将导管插入气管约0.5厘米。确保导管之三未插入太远进气管，因为这会导致肺结构的破坏。 </li><li>通过使用放置在步骤2.1.5棉线绑导管周围气管稳定导管。如果导管没有足够密切，注入平衡盐溶液可以对呼吸道的上部流动，而不是向下进入肺部。 </li></ol></li><li>收集灌洗液<ol><li>加载1mL注射器用1mL与100μMEDTA无菌平衡盐溶液。 </li><li>的1毫升注射器连接到导管并轻轻注入盐/ EDTA溶液进入肺。 </li><li>轻轻吸了解决方案，同时按摩鼠标的胸部。如果吸液的注射器是不可见的，小心地插入导管再稍微向下或向上的气管。 </li><li>从针取下注射器，并将回收的灌洗液转移到15毫升管置于冰上。通常情况下，700 - 900＃181;从1mL注射溶液中回收L的BAL。 </li><li>重复步骤2.2.1 - 2.2.4两次。 注意:如果目的是分析非蜂窝内容，建议在敏感问题时集中样本。 </li></ol></li></ol> 3.收集BAL液体的细胞和非细胞成分<ol><li>在400×g和4℃下离心灌洗液7分钟。 </li><li>收集上清液，并立即用于进一步分析（ 如 ELISA）或在-80°C下冷冻。保持细胞沉淀物分析肺中的细胞流入。 </li><li>将细胞沉淀重悬于200μL的ACK裂解缓冲液中。 注意:此步骤确保红细胞裂解，同时保持白细胞完整。 </li><li>在室温下孵育2分钟。 注意:为了减少由红细胞裂解引起的变异，该步骤不应超过2分钟。 </LI&gt; <li>加入1毫升的冷PBS稀释ACK裂解缓冲液。 </li><li>离心机在400×g离心7分钟，和4℃。弃去上清液并重新悬浮细胞在PBS中的用于下游分析（见下文）的适当体积。 注:PBS的量取决于将要执行的下游研究。 </li></ol> 4.在支气管肺泡灌洗液中不同的细胞类型的流式细胞仪分析注:一种可能性是通过流式细胞术进行流分析BAL流体的绝对和相对的细胞组合物。本文的目的是阐述BAL的技术。流式细胞仪是对自己的一种专门技术。建议以读取流式细胞技术13，14，15，16，17的流动专门论文。抗体与荧光团偶联吨帽子识别特定细胞类型的表面抗原（ 见表1 ）。通过使用门控策略，可以在BAL的细胞部分中鉴定T细胞，巨噬细胞，树突状细胞，B细胞，嗜酸性粒细胞和嗜中性粒细胞。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 抗原 </td><td> 细胞类型 </td></tr><tr><td>分化群3（CD3） </td><td>在T细胞上表达</td></tr><tr><td>分化群11c（CD11c） </td><td>在大多数树突状细胞上高表达，而且在单核细胞，巨噬细胞，嗜中性粒细胞和一些B细胞上表达高。 </td></tr><tr><td>分化簇（CD11b） </td><td>表现为包括单核细胞，嗜中性粒细胞，天然杀伤细胞，粒细胞和巨噬细胞在内的许多白细胞的表面。 </td></tr><tr><td> SiglecF </td><td>一个肺泡巨噬细胞和嗜酸性粒细胞。 </td></tr><tr><td> MHCII </td><td>通常仅在抗原呈递细胞如树突细胞，单核吞噬细胞和B细胞中发现。 </td></tr><tr><td> CD19 </td><td> B淋巴细胞抗原</td></tr><tr><td> LY-6G </td><td>单核细胞，粒细胞和嗜中性粒细胞的标记物</td></tr></tbody></table> 表1:免疫细胞表面抗原的选择。该表提供了用于表征不同细胞类型的表面表位的列表。需要几种标记物的组合来可靠地定义特定的细胞类型。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 样品 </td><td></td><td></td><td></td><td></td></tr><tr><td>管</td><td> 抗原荧光团被添加到细胞中 </TD&gt; <td> 抗体原液浓度（mg / mL）的 </td><td> 抗体稀释 </td><td> 总成交量（微升） </td></tr><tr><td></td><td>可固定活力染料</td><td> 0.2 </td><td> 1/1000 </td><td> 50 </td></tr><tr><td></td><td>表面CD11c </td><td> 0.2 </td><td> 1/800 </td><td> 50 </td></tr><tr><td></td><td> SiglecF </td><td> 0.2 </td><td> 1/100 </td><td> 50 </td></tr><tr><td>样品X </td><td> MHCII </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td></td><td> CD3 </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td></td><td> CD19 </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td></td><td>的CD11b </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td></td><td> Ly6G </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td> 电压控制 </STRONG&gt; </td><td></td><td></td><td></td><td></td></tr><tr><td> 管 </td><td> 抗原-荧光团被添加到细胞 </td><td> 抗体原液浓度（mg / mL）的 </td><td> 抗体稀释 </td><td> 总成交量（微升） </td></tr><tr><td>未染色的细胞</td><td> / </td><td> / </td><td> / </td><td> 50 </td></tr><tr><td>单染色细胞</td><td>可固定活力染料</td><td> 0.2 </td><td> 1/1000 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td>表面CD11c </td><td> 0.2 </td><td> 1/800 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td> SiglecF </td><td> 0.2 </td><td> 1/100 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td> MHCII </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td> CD3 </td><td> 00.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td> CD19 </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td>的CD11b </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td>单染色细胞</td><td> Ly6G </td><td> 0.2 </td><td> 1/200 </td><td> 50 </td></tr><tr><td> 补偿控制 </td><td></td><td></td><td></td><td></td></tr><tr><td> 管 </td><td> 抗原荧光团加入珠粒 </td><td> 抗体库存浓度（mg / mL） </td><td> 抗体稀释 </td><td> 总体积（μL） </td></tr><tr><td>无污染的珠子</td><td> / </td><td> / </td><td> / </td><td> 200 </td></tr><tr><td>单染珠</td><td>表面CD11c </td><td> 0.2 </td><td> 1/2000 </td><td> 200 </TD&gt; </tr><tr><td>单染色珠</td><td> SiglecF </td><td> 0.2 </td><td> 1/2000 </td><td> 200 </td></tr><tr><td>单染色珠</td><td> MHCII </td><td> 0.2 </td><td> 1/200 </td><td> 200 </td></tr><tr><td>单染色珠</td><td> CD3 </td><td> 0.2 </td><td> 1/2000 </td><td> 200 </td></tr><tr><td>单染色珠</td><td> CD19 </td><td> 0.2 </td><td> 1/2000 </td><td> 200 </td></tr><tr><td>单染色珠</td><td>的CD11b </td><td> 0.2 </td><td> 1/400 </td><td> 200 </td></tr><tr><td>单染色珠</td><td> Ly6G </td><td> 0.2 </td><td> 1/200 </td><td> 200 </td></tr></tbody></table> 将包括表2.控制列表。下表显示了所获得的结果的准确解释一切必要的控制。 <ol><li>细胞表面染色 注:这是IMPO包括流式细胞术分析的所有关键控制。需要三套管（ 见表2 ）:（1）含有样品的管; （2）每个抗体荧光团用BAL细胞管制成单一染色体;这允许确定流式细胞仪上每个通道的电压;和（3）每个抗体 - 荧光团的珠子管，以制造单一污渍;这是确定补偿矩阵。 <ol><li>在PBS中以适当的稀释液混合抗体和Fc阻断（抗CD16 / CD32）（ 见表2 ）。在实验前必须确定每种抗体的最佳工作稀释度。 </li><li>将细胞重悬在样品的50μL抗体混合物中，并将50μL适当稀释的抗体加入到关键对照组中。 注意:染色可以在96孔U形板中进行。这使得可以容易地减少染色体积a并运行大量样品。 </li><li>在4℃的黑暗中孵育30分钟。 </li><li>在400×g和4℃下离心7分钟。丢弃上清液。 </li><li>将细胞重新悬浮在PBS中，终体积为200μL。 注意:该最终体积取决于流式细胞仪可以访问的最小体积。机器之间可能有所不同。此外，读取体积取决于样品在流式细胞仪中运行的细胞数量和/或时间。 </li><li>使用样品和对照进行流式细胞分析。 注意:为了确定不同细胞群的绝对细胞数，应加入计数珠。在测量前对每个样品添加相同数量的珠（±25,000珠）。通过使用正向和侧向散射，可以通过流式细胞术鉴定计数珠粒（参见图1 ）。随后，可以通过比较th来计算样本中的绝对数量珠事件细胞事件E比。下列公式可用于: <img alt="方程" src="/files/ftp_upload/55398/55398eq1.jpg" /></li></ol></li><li>流式细胞仪分析 注:流式细胞仪分析，应立即染色方案完成后进行。用适当的激光器和用于信号检测的过滤器流式细胞仪必须使用。 表3给出了所需要的在该手稿中描述的研究中的激光器和过滤器的概述。有关流式细胞仪分析的更多信息，请参阅阿丹等。 18。 <ol><li>建立基于前向散射和侧向散射，不包括碎屑和双峰初级栅极（参见图1）。 </li><li>调整电压和用于与单个染色的细胞和珠的帮助光谱重叠补偿。 注意:这些设置是每个流式细胞仪不同流量和每一个实验前，需要进行检查。 F或正确的流量分析，前向和侧向散射电压是至关重要的。正确的前向和侧向散射可以在鉴定和分析的细胞身份的确认帮助。为了确定这些电压，未染色的样品应先运行。 </li><li>设置荧光门做为表面抗原（ 见图1）和分析样品。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 激光类型 </td><td> 过滤器设置 </td><td></td></tr><tr><td></td><td> 505 LP </td><td>五十〇分之五百二十五</td></tr><tr><td>蓝色（488纳米） </td><td> 550 LP </td><td> 26分之575 </td></tr><tr><td> 100毫瓦</td><td> 670 LP </td><td> 35分之685 </td></tr><tr><td></td><td> 750 LP </td><td>六十零分之七百八十〇 </td></tr><tr><td>紫色405nm的</td><td></td><td> 450/50 </td></tr><tr><td> 100 mW </td><td></td><td></td></tr><tr><td>红色633 nm </td><td></td><td>二十零分之六百六十零</td></tr><tr><td> 70 mW </td><td> 750 LP </td><td>六十零分之七百八十〇 </td></tr></tbody></table> 表3:本研究中使用的流式细胞仪的激光和滤光片概述。

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作者没有什么可以披露的。

BAL是一种有用的技术，以获得细胞学和生物化学信息响应于感染或药物。最初，BAL用于管理从光气中毒3的人类患者的分泌过多粘液。如今，该技术在人类中用于研究肺发病机理，诊断，和疾病3,24治疗管理。在动物实验中，BAL通常用于监测发生在肺气道1,2炎症应答，免疫机制，以及感染性疾病的进程。 为了研究在呼吸系统疾病模型中的炎症细胞的图案，BAL之后应绝对和差分细胞计数。除了绝对细胞数量，相对细胞数量也是令人感兴趣的。例如，维修和癌症模型显示vERY小到无BAL细胞计数升高。在此模型中，细胞组合物的评估是有用的。通过使用细胞染色，在光学显微镜组合，不同的细胞类型，例如嗜酸性粒细胞，嗜中性粒细胞，巨噬细胞和淋巴细胞，可以基于形态25，26，27，28，29，30确定。流式细胞术可用于特定评估，如识别不同的T细胞表型7，31。除了不同的浸润细胞群的鉴定，肺的非细胞组合物可以使用BAL进行调查。方法，如ELISA，免疫印迹，细胞因子微球阵列，免疫组织化学和定量聚合酶链反应是在BAL流体执行，以确定细胞因子，生长因素和其他炎症成分。为了确定肺损伤，BAL液中的总蛋白和乳酸脱氢酶水平也可以被测量32,33 。 随着新的诊断工具的发展，BAL组件的基因组和蛋白质组学特征将在不久的将来成为可能。扩展计算能力和高通量基因表达技术的组合将使得可以定义各种疾病状态的具体基因表达谱。在BAL液上进行这些技术可以提供基因和蛋白质表达模式，以确定参与肺部疾病不同阶段的重要分子。 从BAL流体获得的数据的主要限制是不同研究试验之间缺乏可比性3,9。有很高的程度灌洗技术的变异性和随后的BAL液处理。为了能够比较每个BAL试验，有必要标准化灌注的灌洗液的类型，滴注部位和细胞和非细胞组成要分析的部分。不同试验之间灌洗部分数量存在显着差异，从1到14倍34,35,36不等。这种差异可能对肺中估计的总细胞数有影响。重要的是要知道哪个BAL液体馏分含有大部分的细胞。宋等显示细胞总数约70％以1至3分之22进行检索。然而，其他报告表明，第二次灌洗液比第一次灌洗液中含有更多的细胞37,38 </sup&gt;。我们可以从这些研究得出结论，只有一个部分的灌洗不代表整个肺，导致结果的误解。 BAL液的非细胞组成包含有关33,39,40肺部健康状况的有价值信息。 BAL液稀释的变化有助于可溶性级分的定量差异，从而导致试验结果差异。宋等比较每个灌洗部分的蛋白质和乳酸脱氢酶水平，并得出结论，第一次灌洗部分含有比第二部分多2-3倍。 要检索代表性的BAL样品进行分析，一些技术考虑是至关重要的。其中一个是进行适当的麻醉。检查脚参数非常重要鼠标的lex，以确保终端镇静。这不仅对道德原因很重要，而且因为如果鼠标没有被适当地麻醉，那么难以将导管放置并保持在正确的位置。 第二个重要的技术问题是导管在气管中的位置。当导管插入太深时，可能会损伤肺结构。在BAL过程中，导管的远端不应到达肺部。导管也应稳定并用棉线捆扎。如果导管不稳定，注射的盐水溶液可能向上流入鼻腔而不是向下流入肺。在注射和吸入盐水溶液时，保持导管是很重要的。 从BAL液获得的数据必须代表整个鼠肺。因此，重要的是滴入足够量的盐水缓冲液（ 即3mL，分3份，每份1mL）。细胞产量与BAL液产量之间没有线性关系。在按摩老鼠胸部时轻轻收集溶液是重要的。如果剪切力太强，气道和BAL液中的细胞的活力，功能和结构可能会受到影响。如果吸入的液体在注射器中不可见，请小心地将导管在气管中更深或更高地移动。 应特别注意BAL处理和分析的具体方面。这将最大化BAL样本中保留的信息。在BAL之后，细胞处于营养不良的盐水介质中。因此，在BAL采样后1 h内处理样品非常重要。如果需要长时间的储存，则需要使用补充营养的培养基。 为了保持细胞活力，避免促进细胞粘附于表面的管。避免ce在有可能损害细胞的完整性，或防止所检索的BAL细胞的均匀悬浮速度细胞悬浮液的ntrifugation。 BAL含有流体细胞应在400×g下在4℃下进行7分钟进行离心。重要的是要记住，细胞悬浮液应在4℃下处理期间举行是很重要的。

气道遇到许多侮辱，可能导致炎症，病原体入侵或恶性转化。排列于肺腔的上皮细胞形成哺乳动物身体的主要障碍之一。与肺泡巨噬细胞一起，它们可以防止环境威胁通过气道进入系统系统。这种威胁的例子包括有机和无机化学品，细菌和病毒。同样，可以设计特异性免疫或治疗干预来靶向肺。在所有这些情况下，对诱发反应的详细分析对于理解，干预或预防在呼吸系统内发生的生物过程是重要的。 支气管肺泡灌洗（BAL）是分析这种反应的无价的方法，因为所得样品含有关于炎症反应，免疫机制和感染性疾病进展的重要信息这可能发生在肺气道1,2 中 。通过使用BAL，可以研究渗透细胞。这与消化的肺形成对比，这使得一个"更脏"的细胞群体具有许多死亡和粘性细胞。通过将盐水溶液引入末端细支气管并随后回收该溶液来进行BAL。然后，检索到的溶液可用于定量和表型分析常驻肺和浸润炎症细胞。这种方法经常应用于研究气道疾病模型中的细胞流入，如哮喘，慢性阻塞性肺疾病（COPD）和感染性疾病模型。除了细胞组成外，肺气道的分子组成也反映在BAL液体中。为了分析这一点，酶联免疫吸附测定（ELISA），免疫印迹和通过细胞因子珠阵列同时分析多种细胞因子可以是进行以评估细胞因子和趋化因子的存在。 BAL是研究炎症性炎症性疾病动物模型中炎性细胞的流入的一种已知方法。观察改变的细胞流入（ 例如淋巴细胞，嗜酸性粒细胞或嗜中性粒细胞的水平升高）可以更好地了解疾病，并且可以作为评估治疗干预性能的客观参数。 对BAL细胞分析的准确和可重复的解释需要正确执行BAL，并且收集的流体被正确处理和处理。 Stitt 3在八十多年前引入了"支气管灌洗"这个术语。 1961年，Myrvik从兔肺灌洗液中获得肺泡巨噬细胞3 。 BAL现在是一种常用的分析和监测小鼠模型肺部的方法在科学文献4,5中仍然没有报道标准化的BAL程序。此外，执行BAL可能有很多方法，因为有研究实验室使用技术3,6,7,8,9,10,11。重要的是从BAL获得的数据代表整个鼠肺，而不仅仅是肺的一部分。这种变异使不同试验结果的解释和比较复杂化。 这里描述了一种基本的，便宜的和可重复的BAL方法，其允许收集存在于小鼠的气道腔中的细胞和可溶性级分。简而言之，将导管置于暴露的气管中fa终末麻醉的老鼠。将注射器连接到导管，并将含有乙二胺四乙酸（EDTA）的缓冲盐水溶液引入肺中。使用柱塞通过温和地反复冲洗盐水溶液来取样肺腔。在此步骤中施加的负压最小，以防止气道塌陷。收集后，应进一步处理获得的BAL，以流式细胞计数法细胞计数和鉴定细胞。

<table><tbody><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Thermo Fisher Scientific</td><td>14175-129</td><td></td></tr><tr><td>ethyleendiaminetetra acetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E6511</td><td>irritating</td></tr><tr><td>23 G x 1 1/4 needle</td><td>Henke Sass Wolf</td><td>4710006030</td><td>size: 0.60 x 30 mm</td></tr><tr><td>26 G x 1/2 needle</td><td>Henke Sass Wolf</td><td>4710004512</td><td>size: 0.45 x 12 mm</td></tr><tr><td>plastic tubing</td><td>BD medical technology</td><td>427411</td><td>Polyethylene Tubing, I.D. 0.58 mm (0.023&quot;) O.D. 0.965 mm (0.38&quot;) 30.5 m (100&#039;)</td></tr><tr><td>sodium pentobarbital</td><td>Kela NV</td><td>514</td><td></td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Lonza</td><td>BE17-516F</td><td>PBS without Ca&lt;sup&gt;2+&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt; or phenol red; sterile filtered</td></tr><tr><td>1 mL syringes</td><td>Henke Sass Wolf</td><td>5010.200V0</td><td>nonpyrogenic and nontoxic</td></tr><tr><td>Forceps</td><td>Fine Science Tools GmbH&amp;nbsp;</td><td>91197-00</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools GmbH&amp;nbsp;</td><td>91460-11</td><td></td></tr><tr><td>centrifuge tube 50 mL</td><td>TH.Geyer</td><td>7696705</td><td>Free from Rnase/Dnase/endotoxin</td></tr><tr><td>centrifuge tube 15 mL</td><td>TH.Geyer</td><td>7696702</td><td>Free from Rnase/Dnase/endotoxin</td></tr><tr><td>microcentrifuge tube 1.5 mL</td><td>Sigma-Aldrich</td><td>0030 120.094</td><td>polypropylene</td></tr><tr><td>Microcentrifuge</td><td>Sigma-Aldrich</td><td>5415R</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific</td><td>75004030</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) lysing buffer</td><td>Lonza</td><td>10-548E</td><td>sterile-filtered</td></tr><tr><td>live/dead - efluor 506</td><td>ebioscience</td><td>65-0866-18 &amp;nbsp;</td><td>fixable viability dye</td></tr><tr><td>CD11c-PE-cy7</td><td>eBiosciences</td><td>25-0114-81</td><td></td></tr><tr><td>SiglecF-PE</td><td>BD Pharmingen&amp;nbsp;</td><td>552126&amp;nbsp;</td><td></td></tr><tr><td>MHCII-APCefluor780</td><td>Biolegend</td><td>107628&amp;nbsp;</td><td></td></tr><tr><td>CD3-PE-cy5</td><td>VWR</td><td>55-0031-U100&amp;nbsp;</td><td></td></tr><tr><td>CD19-PE-cy5</td><td>eBiosciences</td><td>15-0193-83&amp;nbsp;</td><td></td></tr><tr><td>CD11b-V450</td><td>BD Pharmingen&amp;nbsp;</td><td>560455&amp;nbsp;</td><td></td></tr><tr><td>Ly6G-AF700</td><td>Biolegend</td><td>127621</td><td></td></tr><tr><td>Absolute Counting Beads</td><td>Life Technologies Europe B.V.&amp;nbsp;</td><td>C36950</td><td></td></tr><tr><td>anti-CD16/CD32</td><td>BD Pharmingen</td><td>553142</td><td></td></tr><tr><td>96-well 340 &amp;micro;l storage plate plate</td><td>Falcon</td><td>353263</td><td>V-bottom, natural polypropylene</td></tr><tr><td>flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>catheter</td><td>BD Biosciences</td><td>393202</td><td></td></tr></tbody></table>

bronchoalveolar lavage of murine lungs to analyze inflammatory cell infiltration

支气管肺泡灌洗（BAL）是一种实验程序，用于离体检查肺腔的细胞和无细胞含量，以了解正在进行的疾病状态。 在这里，描述了一种简单有效的方法来对小鼠肺进行BAL，而不需要特殊的工具或设备。通过将导管插入终末麻醉的小鼠的气管中来分离BAL液，通过该导管将盐水溶液滴入细支气管中。滴入的液体被轻轻地缩回以最大限度地提高BAL液体的回收和最小化剪切力。这种技术允许气道和BAL液中的细胞的存活力，功能和结构被保留。 可以应用许多技术来进一步了解肺的疾病状态。这里，常用的不同类型免疫细胞鉴定和计数的技术是其中流式细胞术与荧光标记的细胞表面特异性标志物的选择组合。本文介绍的BAL程序也可用于分析鼠肺内的感染因子，流体成分或吸入颗粒。

用3×1毫升缓冲盐溶液进行BAL后，2和3毫升之间的容积应该回收。此BAL液可以分析进一步表征的细胞和非细胞的内容。为了研究细胞因子和趋化因子，ELISA 19，免疫印迹20的由细胞因子珠子阵列21的存在，以及多种细胞因子的同时分析可以被执行。此外，该流体的白蛋白和总蛋白质含量可以被确定22。 作为一个例子，本手稿描述如何分析通过流式细胞术BAL液的细胞含量。 24个小时后，他们气管内滴注脂多糖:所分析的BAL液从雌性Balb / cAnNCrl小鼠（7周龄）收集。以下抗体，其耦合到荧光团，瓦特ERE用于识别不同的细胞类型:的CD11c，SiglecF，MHCII，CD3ε，CD19，Ly6g，和CD11b（ 见表1和材料的数据表 ）。可定影活力染料也可使用。通过使用基于所述不同的细胞群体（ 图1）的表面上的抗原的差异表达门控策略，它是可以识别的巨噬细胞，树突细胞，B细胞，T细胞，嗜中性粒细胞和嗜酸性粒。 首先，碎片和双峰基于前向和侧向散射参数门出来。生存力的染料促进了活细胞门控。接下来，CD11c的高细胞和CD11c的低细胞进行鉴定。在的CD11c高人口，巨噬细胞和树突状细胞根据MHCII和SiglecF表达进行了鉴定，分别。在CD11c的低人口，T细胞和B细胞分别基于CD3ε和CD19表达，进行了鉴定。在日基于CD11b和Ly-6G标记表达分别鉴定剩余的细胞群，嗜中性粒细胞和嗜酸性粒细胞。 加入计数珠以通过比较珠粒事件与细胞事件的比例来确定不同细胞群的绝对细胞数23 。这些计数珠基于其前向和侧向散射特性来确定（ 图1 ）。 表4给出了用5μg脂多糖刺激24小时的幼稚小鼠和小鼠的BAL液中不同细胞群的绝对细胞数的概况。 <img alt="图1" src="/files/ftp_upload/55398/55398fig1.jpg" /> 图1:巨噬细胞，树突状细胞，T细胞，B细胞，中性粒细胞和嗜酸性粒细胞i的流式细胞计数检测的门控策略ñBAL液。使用所描述的BAL协议BAL细胞中分离得到。细胞从脂多糖气管内滴注后24个小时的小鼠中分离。算珠和细胞基于前向和侧向散射性质进行鉴定。在单元栅，单细胞使用前向和侧向散射鉴定。在这最后的人口，这是活细胞进行鉴定。那么高的CD11c细胞和CD11c的低细胞进行鉴定。在的CD11c 高人口，巨噬细胞和树突状细胞根据MHCII和SiglecF表达进行了鉴定，分别。在CD11c的低人口，T细胞和B细胞分别基于CD3ε和CD19表达，进行了鉴定。在剩余的细胞群，中性粒细胞和嗜酸性粒细胞分别基于CD11b和Ly的-6G表达，进行了鉴定。 <a href="http://ecsource.jove.com/files/ftp_upload/55398/55398fig1large.jpg" target="_blank">请点击这里查看</a>较大版本的这个数字。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 细胞群体 </td><td> 天真小鼠中绝对数量的细胞 </td><td> LPS刺激小鼠的绝对数量 </td></tr><tr><td> 巨噬细胞 </td><td> 79612 </td><td> 25439 </td></tr><tr><td> 树突细胞 </td><td> 495 </td><td> 671 </td></tr><tr><td> T细胞 </td><td> 45271 </td><td> 28089 </td></tr><tr><td> B细胞 </td><td> 4164 </td><td> 2,926 </td></tr><tr><td> 嗜中性粒细胞 </td><td> 632 </td><td> 566716 </td></tr><tr><td> 嗜酸性粒细胞 </td><td> 3,483 </td><td> 4,332 </td></tr></tbody></table> 表4:代表天真和LPS刺激小鼠肺泡灌洗液流式细胞仪分析结果对准焦点。

Watch this Scientific Journal Video about Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration at JoVE.com

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

肺的健康状况的类型和存在于肺的细支气管的免疫细胞的数目反射。我们描述了支气管肺泡灌洗技术，它允许从小鼠的下呼吸道隔离和非粘附细胞和可溶性因子的研究。

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

bronchoalveolar-lavage-murine-lungs-to-analyze-inflammatory-cell

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

66.8K 次观看.  VIB. 肺的健康状况的类型和存在于肺的细支气管的免疫细胞的数目反射。我们描述了支气管肺泡灌洗技术，它允许从小鼠的下呼吸道隔离和非粘附细胞和可溶性因子的研究。 

视频: Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

科研

免疫与感染

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

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

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

肺环境中免疫细胞和促炎介质的表征

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

在合并臭氧和LPS诱导穆林急性肺损伤期间可视化肺细胞适应

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

无创实时全身体积脉搏波检测小鼠咳嗽敏感性

Methods for Detecting Cough and Airway Inflammation in Mice

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

作者聚焦:调查慢性咳嗽的病理生理学

Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

检测小鼠咳嗽和气道炎症的方法

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge ...

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

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此视频中的章节

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

小鼠与人肺泡巨噬细胞的分离和体外培养

肺环境中免疫细胞和促炎介质的表征

在合并臭氧和LPS诱导穆林急性肺损伤期间可视化肺细胞适应

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

检测小鼠咳嗽和气道炎症的方法

检测小鼠咳嗽和气道炎症的方法