Dye-uptake Experiment through Connexin Hemichannels
连接蛋白半通道染料吸收实验
发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1221 浏览次数: 12108
评审: Vanesa Olivares-IllanaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
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Abstract
Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al., 2013; Tran Van Nhieu et al., 2003). Hemichannel opening can be measured by electrophysiology, by quantifying the release of a hemichannel-permeable molecule into the extracellular medium or by quantifying the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule. As the extent of uptake of a molecule is proportional to its concentration, exposure time, temperature (these parameters are controlled) and, importantly, to the number of active hemichannels on the cell surface, uptake assays are routinely used to assess hemichannel opening. This protocol for the uptake of the fluorescent dye ethidium bromide was used with Hela cells that were stably transfected with Cx26 or Cx43 (Paemeleire et al., 2000). Nevertheless, it could likely be used with other Cx-expressing cell types.
Materials and Reagents
- Cell line of interest and assorted cell culture media and plasticware for propagation from supplier of choice
- DMEM low glucose (Life Technologies, Gibco®, catalog number: A1515401 )
- 100x MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140050 )
- 100x 10,000 U/ml penicillin-streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
- Fetal bovine serum (FBS) (heat-inactivated) (Life Technologies, Gibco®, catalog number: 10500056 )
- Hank’s buffered salt solution (HBSS) (no phenol red) (Life Technologies, Gibco®, catalog number: 14025092 )
- Hank’s buffered salt solution (HBSS) (no calcium, no magnesium, no phenol red) (Life Technologies, Gibco®, catalog number: 14175095 )
- 10x HBSS stock solutions instead of ready to use buffer if desired (Life Technologies, Gibco®, catalog numbers: 14065056 and 14185052 )
- 1 M HEPES (Life Technologies, Gibco®, catalog number: 15630080 )
- 1 M MgCl2
- 0.5 M Na-EGTA
- Ethidium bromide solution (here: eurobio, 0.7 mg/ml)
- Culture medium for propagation of HelaCx26 or HelaCx43 cells (see Recipes)
- HBSS solution (see Recipes)
- HBSS solution devoid of Ca2+ (see Recipes)
- HBSS stock with ethidium bromide (see Recipes)
- Acid washed coverslips (see Recipes)
Equipment
- Incubator for cell culture
- Cover slips and holder or glass bottom dishes for live cell imaging (e.g. MatTek)
- Water bath
- Pipettes
- Aspiration
- Clean bench
- Fluorescence microscope (ideally temperature-controlled) equipped with a camera
Software
- An image analysis software [e.g. ImageJ (http://imagej.nih.gov/ij/) or MetaMorph (Molecular Devices)]
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Puhar, A. and Sansonetti, P. J. (2014). Dye-uptake Experiment through Connexin Hemichannels. Bio-protocol 4(17): e1221. DOI: 10.21769/BioProtoc.1221.
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分类
细胞生物学 > 基于细胞的分析方法 > 转运
细胞生物学 > 细胞成像 > 荧光
生物化学 > 蛋白质 > 结构
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