由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌
Summary
在这篇文章中，我们描述了一种方法，利用多光谱成像流式细胞仪量化酸酐纳米粒子或RAW 264.7细胞细菌的国际化。
Abstract
纳米粒子系统已经出现在疫苗交付有价值的工具，通过他们的能力，有效地交付货物，包括蛋白质，抗原提呈细胞1-5。国际化的纳米粒子的抗原提呈细胞（NP），是在封装的抗原产生有效的免疫反应的关键步骤。为了确定在纳米制定影响功能如何变化，我们寻求开发一种高通量，定量实验的协议，该协议是兼容内在的纳米颗粒以及细菌检测。到今天为止，显微镜和流式细胞仪，两个独立的技术，已用于研究纳米粒子的吞噬功能的方法。流式细胞仪的高通量的性质产生了强大的统计数据。然而，由于分辨率低，不能准确地量化与细胞结合纳米粒子内部。显微镜生成高空间分辨率的图像，H贷款本息偿还期中，它是费时，涉及小样本6-8。多光谱成像流式细胞仪（MIFC）是一种新技术，采用显微镜和流式细胞仪同时执行层的核心，通过多种颜色的光谱荧光和亮场成像方面。此功能提供了一个准确的分析荧光信号强度和高速蜂窝不同的结构和功能之间的空间关系。
在此，我们描述了一个方法，利用MIFC特征的细胞群体的内在酸酐纳米粒子或沙门伤寒。我们还描述了制备纳米粒子悬浮液，细胞标记，ImageStream X系统的采集和分析数据的使用理念的应用。我们也证明了一种技术的应用，可以用来区分国际化带够纳米粒子和细菌athways使用的肌动蛋白介导的吞噬作用的抑制剂细胞松弛素 – D。
Protocol
1。 RAW 264.7巨细胞培养收获RAW 264.7细胞从他们的保温瓶，当他们到达汇合，他们用细胞刮刀轻轻刮。计数和板块他们到1 5 x 10 5细胞密度/以及在完成0.5毫升Dulbecco的修改鹰中等（cDMEM 24孔细胞培养盘; 10％热灭活胎牛血清（FBS），，2毫米Glutamax 10毫米的肝素钠）在5％CO 2培养箱在37℃孵育过夜。 2。致病性沙门氏菌沙门伤寒14028转型与文化<li…
Discussion
有研究表明，聚（乳酸 – 乙醇酸共聚物（PLGA）或酸酐为基础的可生物降解的纳米粒子可以被用于运载包裹抗原或药物靶细胞，其成效是非常重要的吞噬细胞，这些纳米粒子吸收，从而使定量国际化，设计新颖的纳米给药系统的关键，通过使用这种方法。分析，由多种细胞类型的纳米粒子吸收差可以轻松地分析至目前为止，已量化粒子吸收传统的显微镜和流式细胞仪。然而，各自的替代的方法来研…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢金融羚羊的穆里奖（NN00014-06-1-1176）和美国陆军医学研究和装备司令部（批准号W81XWH-09-1-0386和W81XWH-10-1-0806）支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| RAW 264.7 cell line | American Type Culture Collection (ATCC) | TIB-71 | |
| Dulbecco’s Modified Eagle Medium (DMEM) | Cellgro | 10-013-CV | |
| Fetal bovine serum | Atlanta Biologicals | S 11150 | Premium Grade |
| Glutamax | Gibco | 35050-061 | |
| HEPES | Gibco | 15630-080 | |
| 24-well plate | TPP | 92024 | |
| Cell culture Flasks | TPP | 90151 | |
| Cell scraper | TPP | 99002 | 24 cm |
| Salmonella entericaserovar Typhimurium | ATCC | 14028 | |
| BTX ECM630 Electro Cell Manipulator | BTX Harvard Apparatus | ||
| MOPS | Fisher Scientific | BP308 | |
| Phosphate buffered saline (PBS) | Cellgro | 21-040-CV | |
| Ultrasonic liquid processor | Misonix | S-4000 | |
| Cytochalasin-D | Sigma-Aldrich, | C8273 | |
| Formaldehyde | Polysciences | 04018 | |
| Wash buffer | 2% heat inactivated FBS, 0.1% sodium azide in PBS. | ||
| Perm/wash buffer | BD Biosciences | 554714 | |
| Clear-view snap cap microtubes | Sigma | T4816 | |
| Alexa Fluor phalloidin 660 | Invitrogen | A22285 | |
| ImageStreamX | Amnis Corporation | 100200 | Options: 658nm laser, autosampler |
| Sodium azide | Fisher Scientific | S 227I-500 |
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Tags
Cellular InternalizationNanoparticlesBacteriaMulti-spectral Imaging Flow CytometryVaccine DeliveryAntigen Presenting CellsNanoparticle FormulationHigh ThroughputQuantitative Experimental ProtocolMicroscopyFlow CytometryPhagocytosisStatistical DataSpatial ResolutionMulti-color Spectral FluorescenceBright Field Imaging
Cite This Article
Phanse, Y., Ramer-Tait, A. E., Friend, S. L., Carrillo-Conde, B., Lueth, P., Oster, C. J., Phillips, G. J., Narasimhan, B., Wannemuehler, M. J., Bellaire, B. H. Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry. J. Vis. Exp. (64), e3884, doi:10.3791/3884 (2012).