Separation of Intracellular Vesicles for Immunoassays
分离胞内囊泡用于免疫分析
发布: 2015年08月20日第5卷第16期 DOI: 10.21769/BioProtoc.1571 浏览次数: 11961
评审: Jia LiHsin-Yi ChangLee-Hwa Tai
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Sep 2014
Abstract
The endosome/lysosome systems play important roles in immune cell functions as signaling platforms. Immune cells utilize these endosome/lysosome for signal transduction or intercellular communication to elicit the proper immune responses, regulating the localization or the association of the signaling complexes. Here we introduce the procedures to separate the intracellular vesicles such as endosomes or lysosomes, which could be useful to identify the subcellular localization of the signaling complexes.
Materials and Reagents
- Cells (e.g. RAW264.7, Raji, mouse naïve B cells)
- Trypan blue staining (0.4% solution) (Life Technologies, catalog number: 15250-061 )
- CD43-microbeads (Miltenyi Biotec, catalog number: 130-049-801 )
- Blocking One (Nacalai tesque, catalog number: 03953-95 )
- Anti-mouse LAMP1 (eBioscience, catalog number: 1D4B )
- Anti-GM130 (BD biosciences, catalog number: 35/GM130 )
- Anti-Rab5 (Santa Cruz Biotechnologies, catalog number: D-11 )
- Anti-Calnexin (Enzo Life Sciences, catalog number: ADI-SPA-860 )
- Anti-COX IV (Cell Signaling Technology, catalog number: 4844 )
- Anti-rat Ig(H+L)-HRP (Jackson ImmunoResearch Laboratories, catalog number: 712-036-153 )
- Anti-mouse Ig(H+L)-HRP (Jackson ImmunoResearch Laboratories, catalog number: 115-035-062 )
- Anti-rabbit Ig(H+L)-HRP (Jackson ImmunoResearch Laboratories, catalog number: 111-035-144 )
- ECL substrate (Thermo Fisher Scientific, catalog number: 34095 )
- Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 87785 )
- Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: 78420 )
- 60% OptiPrep (Axis-Shield plc, catalog number: 1114542 )
- Solution A (see Recipes)
- Solution B (see Recipes)
- Solution C (see Recipes)
- Solution D (see Recipes)
- Solution E (see Recipes)
- 30%, 23%, 17%, 11%, and 5% Optiprep (2 ml each) (see Recipes)
- TBS-T (see Recipes)
Equipment
- 10 cm dish
- Centrifuge tube (Beckman Coulter, catalog number: 347357 )
- Hemocytometer
- Centrifuge (with cooling function will be recommended)
- 1 ml syringe with 29-gauge needle
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kobayashi, T., Tanaka, T. and Toyama-Sorimachi, N. (2015). Separation of Intracellular Vesicles for Immunoassays. Bio-protocol 5(16): e1571. DOI: 10.21769/BioProtoc.1571.
Download Citation in RIS Format
分类
免疫学 > 免疫细胞分离 > 淋巴细胞
细胞生物学 > 细胞器分离 > 外膜囊泡
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
