Induction of Connexin-hemichannel Opening
Connexin八次跨膜通道白的诱导打开
发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1220 浏览次数: 9033
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). This protocol was used with epithelial cells, in particular with polarized and non-polarized intestinal epithelial TC7 cells and 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. Whether hemichannels are open can be determined by electrophysiology or by measuring the release into the extracellular medium of a hemichannel permeable molecule (for example, ATP) or the uptake of a hemichannel-permeable, plasma membrane-impermeant molecule [for example, the fluorescent dye ethidium bromide-see associated protocol “Dye-uptake Experiment through Connexin Hemichannels” (Puhar and Sansonetti, 2014)].
Keywords: Hemichannel (半通道)
Infection (感染)
Shigella (志贺菌)
Salmonella (沙门氏菌)
Enteropathogenic Escherichia coli (EPEC) (致病性大肠杆菌（EPEC）)
Materials and Reagents
- Cell line of interest and assorted cell culture media and plasticware for propagation from supplier of choice
- Shigella flexneri (wild-type or mutant of interest) expressing the adhesin AfaE [for example, serotype 5a wild-type strain M90T, serotype 5a non-invasive mxiD- mutant (for negative controls), or serotype 5a ipgD- mutant (upon infection with this strain hemichannels will stay open for extended time)]
- Salmonella enterica serovar Typhimurium (for example wild-type strain SL1344)
- Enteropathogenic Escherichia coli (EPEC) (for example serotype O127: H6 wild-type strain E2348/69)
- Biocoat intestinal epithelium differentiation environment (BD Biosciences, catalog number depends on well size)
- 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 HBSS 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
- Growth media for bacteria [for example Luria-Bertani broth (Life Technologies, catalog number: 10855001 ); Luria-Bertani agar powder prepared according to manufacturer’s instructions (Life Technologies, catalog number: 22700025 ) for Salmonella and EPEC; tryptic soy broth (soybean-casein digest medium, BD, catalog number: 257107 ); tryptic soy agar powder prepared according to manufacturer’s instructions (soybean-casein digest agar medium, Difco, catalog number: 236950 ) for Shigella]
- Growth media for bacteria containing Congo red (see Recipes)
- Culture medium for propagation of TC7 cells (see Recipes)
- Culture medium for propagation of HelaCx26 or HelaCx43 cells (see Recipes)
- HBSS solution (see Recipes)
- HBSS solution devoid of Ca2+ (see Recipes)
Equipment
- Incubator for cell culture
- Cover slips of desired size (e.g. 24 well plates) (if desired)
- Water bath
- Pipettes
- Aspiration
- Clean bench
- Incubator for bacterial cultures
- Centrifuge adapted to spin culture plates (for infections with EPEC)
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). Induction of Connexin-hemichannel Opening. Bio-protocol 4(17): e1220. DOI: 10.21769/BioProtoc.1220.
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分类
微生物学 > 微生物-宿主相互作用 > 体外实验模型 > 细胞系
微生物学 > 微生物生物化学 > 蛋白质 > 活性
细胞生物学 > 基于细胞的分析方法 > 转运
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