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
Summary
我们说明进入肺部通过气管内麻醉小鼠的非手术治疗提供试验材料。这种方法允许肺暴露于细菌和病毒的病原体，细胞因子，抗体，珠子，化学品，染料。我们进一步描述采伐和肺和肺淋巴结（LDLNs），流式细胞仪检测处理。
Abstract
连续采样在肺部的肺泡吞噬细胞，如肺泡巨噬细胞和肺树突状细胞的最不发达国家（LDCs）的抗原。最不发达国家，特别是被称为迁移到肺淋巴结（LDLNs）他们目前吸入各种免疫原1,2发起一个适当的免疫反应的T细胞抗原。肺和空气中的抗原在小鼠模型之间的相互作用，抗原滴鼻给药1,3,4，气管或气溶胶6。每条路线交货涉及不同的技术技能和限制，需要考虑设计一个实验之前。例如，鼻腔和雾化曝光提供抗原肺部和上呼吸道。因此，抗原可进入鼻相关淋巴组织（NALT）7，潜在的复杂结果的解释。此外，吞咽，打喷嚏和鼠标的呼吸频率，也可能导致在交付的剂量不一致。虽然上呼吸道的参与可能是一些研究的首选，它可以实验侧重于专门在肺部发起的事件复杂化。在这种背景下的气管（IT）的路线是可取的，因为它提供的测试材料，直接进入肺部，并绕过NALT。许多注射协议涉及要么盲目通过口腔或气管的手术暴露访问肺部气管插管。在这里，我们描述了一个简单的，一致的，它滴入的非手术方法。气管的开放是可视化使用喉镜和弯曲灌胃针插入进气管直接提供innoculum的。我们还描述了采伐和加工用流式细胞仪分析抗原贩运LDLNs和肺部的程序。
Protocol
1。之前的过程中，准备和收集下列项目请参考图1a中的形象）和1b为建设一个木制的平台，以抑制在操作过程中鼠标）。 消化混合淋巴结 – 的HBSS + 1.25mg/ml胶原酶TypeIV或2.5 mg / mL胶原酶ð 消化肺部混合 – 的HBSS + 1 mg / mL胶原酶稀释在PBS腹腔注射乳胶珠（1:20）的LDLNs可视化 注：LDLNs很小，很难找到。要了解如何找到他们，注入200μL稀释?…
Discussion
我们利用这个协议，从肺到LDLNs 研究炭疽杆菌孢子的贩运。对于类似的应用程序，传送到肺部的颗粒数应慎重选择，使注射材料可通过流式细胞仪检测的LDLNs。我们还成功地使用这种方法为具体的肺细胞用荧光标记抗体的人口的细胞和标签的收养转移。此外，这种方法是经常使用的管理直接进入肺部10博莱霉素（化学诱导的肺间质纤维化）和细胞因子。
尽管此?…
Disclosures
The authors have nothing to disclose.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| 70μm Cell stariner | BD | ||
| Aniline Blue | Fisher | A967-25 | |
| Animal Feeding needle | Popper and Sons Inc | 7920 | |
| Collagenase | Sigma | C2139 | |
| Collagenase TypeIV | Worthington | ||
| Collagenase D | Roche | 11088974103 | |
| DPBS | Invitrogen | 14190 | |
| Fluoresbrite YG Microspheres (0.5μm) | Polysciences, Inc | 17152 | |
| HBSS without Calcium chloride and magnesium chloride | Invitrogen | 14170 | |
| Ketamine HCl (100mg/ml) | Hospira Inc | ||
| Laryngoscope Blade | PennCentury, Inc | For Model LS-1 | Refer to www.penncentury.com |
| Lightweight Fiber Optic Laryngoscope | WelchAllyn | 80814 | |
| Red Fluorescent Beads (0.5μm) | Invitrogen | F8812 | For i.p injection |
| Xylazine (100mg/ml) | Lloyd Laboratories |
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Tags
Non-surgicalIntratracheal InstillationMiceAnalysisLungsLung Draining Lymph NodesFlow CytometryPhagocytic CellsAlveolar MacrophagesLung Dendritic CellsAntigensImmune ResponseInhaled AntigensT CellsIntranasal AdministrationAerosolized ExposureTechnical SkillsLimitationsUpper Respiratory TractInterpretation Of ResultsSwallowingSneezingBreathing Rate Of The MouseDoses DeliveredI.t RouteTest MaterialsBlind Intubation
Cite This Article
Rayamajhi, M., Redente, E. F., Condon, T. V., Gonzalez-Juarrero, M., Riches, D. W., Lenz, L. L. Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry. J. Vis. Exp. (51), e2702, doi:10.3791/2702 (2011).