Differentiation of Naturally Produced Extracellular Membrane Vesicles from Lipid Aggregation by Glucuronoxylomannan Immunogold Transmission Electron Microscopy in Bacillus subtilis
采用Glucuronoxylomannan免疫胶体金透射电子显微镜法检测枯草杆菌中脂质聚合自然产生的胞外膜囊泡的分化
发布: 2015年03月05日第5卷第5期 DOI: 10.21769/BioProtoc.1408 浏览次数: 10515
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jul 2014
Abstract
Recently, membrane vesicle (MV) production was described in Gram-positive bacteria, which harbor a variety of components such as toxins, antibiotic resistance proteins, proteases, DNA, and immune modulators. Free lipids have the ability to form micelles, thus it is important to rule out spontaneous association of lipids into vesicle-like structures and rather, that MVs are produced naturally by a metabolically active cell. Here, we describe a protocol utilizing the polysaccharide, glucuronoxylomannan (GXM) from Cryptococcus neoformans (C. neoformans) as a marker to differentiate naturally produced MVs from vesicles that form spontaneously in the Gram-positive model organism, Bacillus subtilis (B. subtilis). MVs are purified from bacterial cultures grown in the presence of GXM; MVs naturally produced by cells would not contain GXM in the lumen whereas vesicular structures forming in the media could encapsulate GXM and this can be visualized via immunogold transmission electron microscopy.
Materials and Reagents
- Purified glucuronoxylomannan (GXM) (protocol included)
- YPD broth (Difco, catalog number: 242820 )
- C. neoformans H99 fungal strain
- 100 kDa, 50 kDa, 10 kDa, 1 kDa cut-off EMD Millipore Ultrafiltration Membranes (Millipore, catalog number: 14442AM )
- B. subtilis 168 bacterial strain
- BHI broth (Difco, catalog number: 299070 )
- 8% glutaraldehyde (Polysciences, catalog number: 111-30-8 )
- 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15700 )
- 0.5 M sodium cacodylate (pH 7.4) (Electron Microscopy Sciences, catalog number: 11650 )
- 100% ethanol
- Lowicryl HM-20 monostep resin (Electron Microscopy Sciences, catalog number: 14340 )
- Gelatin (Thermo Fisher Scientific)
- Aurion Donkey Block (Electron Microscopy Sciences, catalog number: 25599 )
- α-GXM monoclonal antibody 18B7 (mouse monoclonal IgG1) (Casadevall lab-generated)
- BSA-c (Electron Microscopy Sciences, catalog number: 25557 )
- 10 nm conjugated secondary Ab (donkey α-mouse) (Electron Microscopy Sciences, catalog number: 25814 )
- 4% uranyl acetate (aq) (SPI Supplies, catalog number: 615-44-0 )
- 1x phosphate buffered saline (PBS) (see Recipes)
Equipment
- Express PLUS Membrane Filters - Pore (0.22 μm) (Millipore, catalog number: SCGVU01RE )
- Ultracentrifugation tubes (thickwall, polyallomer/pollypropylene, 3.5 ml, 13 x 51 mm) (Beckman Coulter, catalog number: 349623 )
- 200 mesh nickel grids (Polysciences, catalog number: 24916 )
- Centrifuge capable of 15,000 x g
- TLA 100.3 rotor (Beckman Coulter, catalog number: 349490 )
- Amicon ultrafiltration system (Millipore, catalog number: 5124)
- Optima TL ultracentrifuge (Beckman Coulter, discontinued comparable to B11229)
- Sonicator (sonic dismembrator) (Thermo Fisher Scientific, model: 100 cpn-214-161 )
- Freeze substitution system (RMC, model: FS-7500 )
- Reichert Ultracut UCT Ultramicrotome
- JEOL 100CXII or JEOL 1200EX Electron Microscopes
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Brown, L., Perumal, G. and Casadevall, A. (2015). Differentiation of Naturally Produced Extracellular Membrane Vesicles from Lipid Aggregation by Glucuronoxylomannan Immunogold Transmission Electron Microscopy in Bacillus subtilis. Bio-protocol 5(5): e1408. DOI: 10.21769/BioProtoc.1408.
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分类
微生物学 > 微生物细胞生物学 > 细胞成像
微生物学 > 微生物细胞生物学 > 细胞器分离
细胞生物学 > 细胞成像 > 电子显微镜
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