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
Summary
在此，我们描述了小鼠肺泡巨噬细胞，居住在肺先天免疫细胞，并检查其激活，共培养酸酐纳米收获的协议。
Abstract
可生物降解的纳米粒子已经出现作为一个通用平台的设计和实施新鼻内疫苗对呼吸道传染病。具体来说，酸酐纳米脂肪癸二酸（SA），芳香1,6 -双（P-羧基苯氧）正己烷（CPH），或双亲1,8 -双（P-羧基苯氧）-3,6 – dioxaoctane组成（CPTEG）显示独特的体积和表面侵蚀动力学1,2和可利用缓慢释放功能的生物大分子（如蛋白抗原，免疫球蛋白等） 在体内 3,4,5。这些纳米粒子也具有内在的辅助活动，使他们6,7,8疫苗交付平台的一个很好的选择。
为了阐明机制激活先天免疫后鼻腔粘膜接种，必须评估的分子和细胞反应的抗原P怨恨细胞（APC）负责启动免疫反应。树突状细胞是进行气管中发现的主要装甲运兵车，而在肺实质9,10,11（AMɸ）肺泡巨噬细胞为主。 AMɸ是非常有效地清除病原微生物和细胞碎片12,13肺部。此外，这种细胞类型发挥宝贵的作用在运输微生物抗原的淋巴结，这是一个重要的第一步启动适应性免疫反应9。 AMɸ也表达水平升高的先天模式识别和清道夫受体，分泌促炎介质，和总理幼稚T细胞12,14。一个人口相对纯的AMɸ（例如，大于80％），可以很容易地获得通过肺灌洗在实验室中研究。从免疫动物AMɸ收获的居民提供了一个代表性的表型的巨噬细胞，将encounter的粒子在体内的疫苗。在此，我们描述了从小鼠到收获和文化AMɸ使用的协议和检查酸酐纳米粒子在体外治疗后的巨噬细胞的激活型。
Protocol
1。鼠标采用肺灌洗收获（AMɸ）肺泡巨噬细胞穿白大褂，一次性手套，和适当的保护眼睛，如适当的个人防护装备（PPE）。 开始收获前准备完整AMɸ（cAMɸ）培养基。新增2.5青霉素/链霉素（笔/链球菌）解决方案，250β-巯基乙醇微升（巯基乙醇）和25热灭活胎牛血清（FBS），毫升222.25 Dulbelcco的修改鹰培养基（DMEM培养基）毫升毫升。过滤消毒cAMɸ介质在真空条件下使用0.2微米的孔大小瓶…
Discussion
酸酐纳米疫苗平台显示效果时，给予单剂量疗法5滴鼻。在此疫苗交付平台引起的肺部测量驻地吞噬细胞群的激活，允许评估其潜在的能力，最终促进适应性免疫反应。
具体来说，从肺灌洗液中收获肺泡巨噬细胞和不同配方的纳米粒子治疗提供了不同的粒子化学能力的见解，激活巨噬细胞，导致抗原提呈6,8。此外， 在体外研究中，这些都是用于?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者想感谢来自衣阿华州立大学流式细胞设施美国陆军医学研究和装备司令部（格兰特W81XWH-09-1-0386和W81XWH-10-1-0806号）的财政支持和肖恩·里格比博士他的专家技术援助。
Materials
| Name of the reagent | Company | Catalog number | Comments |
| cAM Media | |||
| DMEM | Cellgro | 15-013-CV | |
| 50 mM 2-mercaptoethanol | Sigma | M3148-25ML | |
| Penicillin/Streptomycin 10,000 μg/ mL Solution | Cellgro | 30-002-CI | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150 | |
| FACS Buffer | |||
| Sodium chloride | Fisher Scientific | S671-500 | |
| Sodium phosphate | Fisher Scientific | MK7868500 | |
| Potassium chloride | Fisher Scientific | P217500 | |
| Potassium phosphate | Fisher Scientific | P288-200 | |
| BSA (Bovine Serum Albumin) | Sigma | A7888 | |
| Sodium Azide | Sigma | S2002 | |
| Antibodies | |||
| Rat IgG | Sigma | I4341 | |
| Anti-Ms CD16/32 | eBioscience | 16-0161 | |
| Anti-Ms MHC II haplotype I-A/I-E, clone M5/114.15.2, conjugated to fluorescein isothiocyanate (FITC) | eBioscience | 11-5321 | |
| Anti-mouse CD86, clone GL-1, conjugated to allophycocyanin (APC)-Cy7 | Biolegend | 105030 | |
| Anti-mouse CD40, clone 1C10, conjugated to APC | eBioscience | 17-0401 | |
| Anti-mouse CD209, clone 5H10, conjugated to Biotin | eBioscience | 13-2091 | |
| Anti-mouse CD11b, clone M1/70, conjugated to Alexa Fluor 700 | eBioscience | 56-0112 | |
| Anti-mouse F4/80, clone BM8, conjugated to phycoerythrin (PE)-Cy7 | eBioscience | 25-4801 | |
| PE-Texas red conjugated Streptavidin | BD Biosciences | 551487 | |
| Other Supplies and Reagents | |||
| Ethanol | Fisher Scientific | A405-20 | Used as 70% (v/v) |
| Compressed CO2 | Linweld | 16000060 | |
| 1 mL Syringe | BD Biosciences | 309659 | |
| Sovereign 3 ½” Fr Tom Catcatheter | Kendall | 703021 | |
| Biosafety Cabinet | NUAIRE | Series 22 | |
| Dissection Scissors | Fisher | 138082 | |
| Forceps | Roboz | RS-8254 | |
| PBS, 1X without calcium and magnesium | Cellgro | 21-040-CM | |
| 15 mL Centrifuge Tubes with Screw Cap | VWR International | 21008-216 | |
| Six-well Tissue Culture Treated Plates | Costar | 3516 | |
| Plastic Tube Racks | Nalgene | 5970 | |
| Cell Scraper 24 cm | TPP | 99002 | |
| 5 mL Polystyrene Round-Bottom Tube | Falcon | 352008 | |
| Pipet-aid XL | Drummond | 4-000-105 | |
| 10, 5, and 2 mL Pipettes | Fisher | 13-675 | |
| 200 and 10 μL micropipettors | Gilson Pipetman | F123601 | |
| 200 and 10 μL pipette tips | Fisher | 02-707 | |
| BD Stabilizing Fixative | BD Biosciences | 338036 | |
| Isoton II Diluent | Beckman-Coulter | 8546719 | |
| Zap-oglobin II Lytic Reagent | Beckman-Coulter | 7546138 | |
| Coulter Counter Polystyrene Vials | Beckman-Coulter | 14310-684 | |
| Test Tubes | BD Biosciences | 352008 | |
| Equipment | |||
| Refrigerated Centrifuge | Labnet | 50075040 | |
| Humidified Incubator CO2 | Nuaire | Model Autoflow 8500 | |
| FACSCanto Flow Cytometer | BD Biosciences | 338960 | |
| Coulter Particle Counter Z1 | Beckman-Coulter | WS-Z1DUALPC | |
| Sonicator Liquid Processing Equipment with Microtip | Misonix | Model No. S-4000 |
References
- Mallapragada, S. K., Narasimhan, B. Immunomodulatory biomaterials. Int. J. Pharm. 364, 265-271 (2008).
- Torres, M. P., Vogel, B. M., Narasimhan, B., Mallapragada, S. K. Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms. J. Biomed. Mater. Res. A. 76, 102-110 (2006).
- Lopac, S. K., Torres, M. P., Wilson-Welder, J. H., Wannemuehler, M. J., Narasimhan, B. Effect of polymer chemistry and fabrication method on protein release and stability from polyanhydride microspheres. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 91, 938-947 (2009).
- Torres, M. P., Determan, A. S., Anderson, G. L., Mallapragada, S. K., Narasimhan, B. Amphiphilic polyanhydrides for protein stabilization and release. Biomaterials. 28, 108-116 (2007).
- Ulery, B. D. Design of a Protective Single-Dose Intranasal Nanoparticle-Based Vaccine Platform for Respiratory Infectious Diseases. PLoS ONE. 6, e17642 (2011).
- Petersen, L. K., Xue, L., Wannemuehler, M. J., Rajan, K., Narasimhan, B. The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells. Biomaterials. 30, 5131-5142 (2009).
- Petersen, L. K. Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants. Biomaterials. 32, 6815-6822 (2011).
- Torres, M. P. Polyanhydride microparticles enhance dendritic cell antigen presentation and activation. Acta. Biomater. 7, 2857-2864 (2011).
- Kirby, A. C., Coles, M. C., Kaye, P. M. Alveolar macrophages transport pathogens to lung draining lymph nodes. J. Immunol. 183, 1983-1989 (2009).
- Barletta, K. E. Leukocyte compartments in the mouse lung: Distinguishing between marginated, interstitial, and alveolar cells in response to injury. J. Immunol. Methods. , (2011).
- Rubins, J. B. Alveolar macrophages: wielding the double-edged sword of inflammation. Am. J. Respir. Crit. Care Med. 167, 103-104 (2003).
- Sun, K., Gan, Y., Metzger, D. W. Analysis of Murine Genetic Predisposition to Pneumococcal Infection Reveals a Critical Role of Alveolar Macrophages in Maintaining the Sterility of the Lower Respiratory Tract. Infect. Immun. 79, 1842-1847 (2011).
- Morimoto, K. Alveolar Macrophages that Phagocytose Apoptotic Neutrophils Produce Hepatocyte Growth Factor during Bacterial Pneumonia in Mice. Am. J. Respir. Cell Mol. Biol. 24, 608-615 (2001).
- Gordon, S., Taylor, P. R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953-964 (2005).
- Ulery, B. D. Polymer chemistry influences monocytic uptake of polyanhydride nanospheres. Pharm. Res. 26, 683-690 (2009).
- Petersen, L. K., Sackett, C. K., Narasimhan, B. High-throughput analysis of protein stability in polyanhydride nanoparticles. Acta Biomater. 6, 3873-3881 (2010).
- Irvin, C. G., Bates, J. H. Measuring the lung function in the mouse: the challenge of size. Respir. Res. 4, 4 (2003).
Tags
Murine Alveolar MacrophagesCellular ActivationPolyanhydride NanoparticlesIntranasal VaccinesRespiratory Infectious DiseasesSebacic Acid16-bis(p-carboxyphenoxy)hexane18-bis(p-carboxyphenoxy)-36-dioxaoctaneBulk Erosion KineticsSurface Erosion KineticsBiomolecules ReleaseAdjuvant ActivityVaccine Delivery PlatformInnate ImmunityMolecular ResponsesCellular ResponsesAntigen Presenting Cells (APCs)Dendritic CellsAlveolar Macrophages (AM)Lung ParenchymaMicrobial PathogensCell Debris
Citer Cet Article
Chavez-Santoscoy, A. V., Huntimer, L. M., Ramer-Tait, A. E., Wannemuehler, M., Narasimhan, B. Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles. J. Vis. Exp. (64), e3883, doi:10.3791/3883 (2012).