Functional Analysis of Connexin Channels in Cultured Cells by Neurobiotin Injection and Visualization
通过神经生物素注射和可视化对培养细胞中连接蛋白通道进行功能分析
发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2325 浏览次数: 9038
评审: Andrea GramaticaGunjan MehtaAnonymous reviewer(s)
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Apr 2017
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Abstract
Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured cells.
Keywords: Gap junction (间隙连接)
Connexin (连接蛋白)
Neurobiotin (神经生物素)
Microinjection (显微注射)
Iontophoresis ( 离子电渗疗法)
Background
Gap junctions are intercellular conduits formed between neighboring cells, allowing the diffusional exchange of low molecular mass molecules (< 1.8 kDa). A gap junction channel consists of two hemichannels (connexons) docked to each other. Each connexon is a hexameric assembly of protein subunits termed connexins (Cx). The gap junction protein gene family consists of 20 members in mice and 21 in humans. Connexins are named according to their approximate molecular mass in kDa e.g., Cx43 has an approximate molecular mass of 43 kDa (for review see Söhl and Willecke, 2004). Different connexins are widely expressed in a variety of tissues throughout development where they mediate electrical as well as metabolic coupling. Furthermore, second messenger molecules and ions can be exchanged by direct diffusion through gap junctional channels.
Neurobiotin (N-(2-aminoethyl)biotinamide) is a compound of 286 Da molecular mass and a charge of +1 under physiological conditions. Due to its small size, this tracer passes even those gap junction channels which are not permeable to other common tracers of higher molecular mass e.g., Lucifer Yellow or carboxyfluorescein (Hampson et al., 1992), therefore representing a very sensitive method to detect gap junctional intercellular communication. Compared to the similar tracer biocytin, Neurobiotin appears to be superior regarding solubility, and stability. Furthermore, the compound can be selectively iontophoresed with positive current and subsequently fixed using paraformaldehyde or glutaraldehyde (Kita and Armstrong, 1991). As Neurobiotin does not show autofluorescence it needs to be detected using Avidin conjugated either to horseradish peroxidase or directly linked to a fluorescent dye.
Materials and Reagents
- 6 cm tissue culture treated culture dishes (several distributers available e.g., Corning, Tewksbury, MA)
- GB 100-F8P borosilicate glass capillaries (Science Products GmbH, Hofheim, Germany)
- Syringe (1 ml)
- Spinal needle (0.5 mm, 25 G)
- Reaction tube
- Cell line or primary cell preparation of interest
- pcDNATM3.1/Zeo(+)
- HistoGreen HRP-substrate Kit (Linaris, catalog number: E109 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
- Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
- Neurobiotin (Vector Laboratories, catalog number: SP-1120 )
- Rhodamine B isothiocyanate-Dextran (Sigma-Aldrich, catalog number: R9379 )
- Tris base (Sigma-Aldrich, catalog number: T1503 )
- Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: L9650 )
- 50% glutaraldehyde solution (Sigma-Aldrich, catalog number: 340855 )
- Triton X-100 (Sigma-Aldrich, catalog number: X100 )
- Avidin D coupled horseradish peroxidase (Vector Laboratories, catalog number: A-2004 )
- PBS (see Recipes)
- Neurobiotin/Rhodamine B isothiocyanate-Dextran solution (see Recipes)
- LiCl solution (see Recipes)
- 0.5% glutaraldehyde solution (see Recipes)
- Triton X-100 solution (see Recipes)
- Avidin D coupled horseradish peroxidase solution (see Recipes)
Equipment
- Microelectrode-holder suitable for 1 mm glass capillaries with AgCl electrode (e.g., World Precision Instruments, model: MEH8 )
- Dual Microiontophoresis current generator SYS-260 (World Precision Instruments, FL)
- Zeiss IM35 inverted fluorescence microscope (Zeiss, model: Zeiss IM35 ) equipped with:
- Heated stage set to 37 °C
- HBO lamp (100 W)
- Appropriate filter set to detect Rhodamine B fluorescence (Ex/Em 570/590; Filter Set 20 HE)
- The microscope should be placed on an anti-vibration microscope desk
- Incubator
- Micropipette puller P97 (Sutter Instrument, model: P-97 )
- Micromanipulator Injectman (Eppendorf, Hamburg, Germany)
- AgCl reference electrode (disc)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wörsdörfer, P. and Willecke, K. (2017). Functional Analysis of Connexin Channels in Cultured Cells by Neurobiotin Injection and Visualization. Bio-protocol 7(11): e2325. DOI: 10.21769/BioProtoc.2325.
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分类
干细胞 > 成体干细胞 > 维持和分化
细胞生物学 > 细胞成像 > 荧光
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