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Dye-uptake Experiment through Connexin Hemichannels
连接蛋白半通道染料吸收实验   

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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

  1. Cell line of interest and assorted cell culture media and plasticware for propagation from supplier of choice
  2. DMEM low glucose (Life Technologies, Gibco®, catalog number: A1515401 )
  3. 100x MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140050 )
  4. 100x 10,000 U/ml penicillin-streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
  5. Fetal bovine serum (FBS) (heat-inactivated) (Life Technologies, Gibco®, catalog number: 10500056 )
  6. Hank’s buffered salt solution (HBSS) (no phenol red) (Life Technologies, Gibco®, catalog number: 14025092 )
  7. Hank’s buffered salt solution (HBSS) (no calcium, no magnesium, no phenol red) (Life Technologies, Gibco®, catalog number: 14175095 )
  8. 10x HBSS stock solutions instead of ready to use buffer if desired (Life Technologies, Gibco®, catalog numbers: 14065056 and 14185052 )
  9. 1 M HEPES (Life Technologies, Gibco®, catalog number: 15630080 )
  10. 1 M MgCl2
  11. 0.5 M Na-EGTA
  12. Ethidium bromide solution (here: eurobio, 0.7 mg/ml)
  13. Culture medium for propagation of HelaCx26 or HelaCx43 cells (see Recipes)
  14. HBSS solution (see Recipes)
  15. HBSS solution devoid of Ca2+ (see Recipes)
  16. HBSS stock with ethidium bromide (see Recipes)
  17. Acid washed coverslips (see Recipes)

Equipment

  1. Incubator for cell culture
  2. Cover slips and holder or glass bottom dishes for live cell imaging (e.g. MatTek)
  3. Water bath
  4. Pipettes
  5. Aspiration
  6. Clean bench
  7. Fluorescence microscope (ideally temperature-controlled) equipped with a camera

Software

  1. An image analysis software [e.g. ImageJ (http://imagej.nih.gov/ij/) or MetaMorph (Molecular Devices)]

Procedure

  1. Cells were grown at 37 °C and 10% CO2 in a humid environment (incubator) and passaged according to standard protocols.
  2. Cells were seeded on acid-washed cover slips of desired size (e.g. 24 well plates) (see Recipes) or glass-bottom dishes the day before the experiment and used at 70 % confluency.
  3. A stock solution of HBSS devoid of Ca2+ containing ethidium bromide (e.g. 5 μM) was prepared to make sure that all samples were exposed to exactly the same concentration of dye.
  4. Cells were carefully washed twice with warm (37 °C) HBSS and once with warm HBSS devoid of Ca2+. For negative controls, only HBSS was used. To wash, carefully aspirate the medium or buffer, replace with fresh buffer, carefully pipet up and down once or twice, and replace with fresh buffer.
  5. Cells were loaded with pre-warmed 5 μM ethidium bromide in HBSS devoid of Ca2+ (to induce hemichannel opening) for 10 min at 37 °C. For negative controls pre-warmed 5 μM ethidium bromide in HBSS containing Ca2+ was used.
  6. The supernatant was discarded and cells washed with warm HBSS containing Ca2+ to induce hemichannel closure and thereby trap the dye in the cytosol.
  7. Images were acquired within 30 min (the ethidium bromide signal was stable during this period) at 37 °C and 40x magnification. Make sure enough separate fields within a sample are acquired and prepare sufficient independent repeats for quantification. Please refer to Notes for the settings of the fluorescence microscope.
  8. Intracellular ethidium bromide-fluorescence of single cells was quantified using an image analysis software. The background fluorescence was subtracted for every field.

Representative data

  1. Representative result


    Figure 1. Typical results for the assay performed on HelaCx26 cells. Uptake of ethidium bromide was quantified by fluorescence and values were normalized to those of positive controls (in which hemichannel opening was induced by low extracellular Ca2+), which were set to 100. In negative controls hemichannel opening was not induced, hence, the background fluorescence corresponds mostly to ethidium bromide taken up by endocytosis. The mean with SD of approximately 900 cells from three independent experiments are shown.18α-glycyrrhetinic acid (AGA), 100 μM (a hemichannel inhibitor).


    Figure 2. Typical appearance of HelaCx26 cells loaded with ethidium bromide. Cells were exposed for 10 min to ethidium bromide in HBSS devoid of Ca2+ to induce hemichannel opening (this type of sample corresponds to a positive control in Figure 1). Fluorescent A and transmission B images of cells are shown. Dying cells and debris (highlighted by red rectangles in B) should be omitted for quantification. The compromised plasma membrane permeability of dying cells will allow non-hemichannel-dependent passage of ethidium bromide. Ethidium bromide fluorescence is enhanced when the dye is bound to DNA.

  2. Notes about reproducibility and variability in results
    Variability is low (10%), as dye uptake is very homogenous with a population of cells. There is an about two to three-fold difference between positive and negative controls. Shorter ethidium bromide pulses result in higher variability (due to differences in handling time), but also slightly higher differences between positive and negative controls, because less ethidium bromide is taken up by endocytosis.

Notes

  1. Please also refer to the protocol “Induction of Connexin-hemichannel Opening” described by Puhar and Sansonetti (2014).
  2. Many cell lines have lost expression of Cxs and therefore do not have hemichannels. Make sure your cell line of interest does.
  3. During the experiment, handle cells carefully, as hemichannels are mechanosensitive. Excessive shaking and vigorous exchange of media will induce hemichannel opening and therefore increase the background for subsequent measurements.
  4. For HBSS, 10x stock solutions may be used.
  5. Handle ethidium bromide with care, as it’s a DNA-intercalating agent, and dispose of ethidium bromide-containing solutions in dedicated containers.
  6. For single-cell experiments, quantification from microscope images are mandatory. However, if bulk measurements are desired, a fluorescence plate-reader can be used instead of a microscope.
  7. If higher concentrations of ethidium bromide are used, incubation times should be reduced. However, with very short incubation times differences in handling times will make the comparison of samples more difficult.
  8. The excitation and emission maxima of ethidium bromide are 524 nm and 605 nm, respectively. Hence, for ethidium bromide green excitation filters and orange emission filters should be used. Typically, standard microscope settings for Cy3/TRITC/rhodamine can be used, but for optimal performance the best combinations of available filters should be determined by the experimenter.

Recipes

  1. Culture medium for propagation of HelaCx26 or HelaCx43 cells
    Mix 500 ml sterile DMEM with sterile 5 ml penicillin + streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin final, optional) and 50 ml sterile FBS
    Stored at 4 °C
  2. HBSS solution
    Mix HBSS with 1M HEPES to final concentration of 20 mM (e.g. 1 ml 1 M HEPES for 50 ml final volume)
  3. HBSS solution devoid of Ca2+
    Mix HBSS devoid of calcium and magnesium with 1 M HEPES to final concentration of 20 mM (e.g. 1 ml 1 M HEPES for 50 ml final volume), MgCl2 to final concentration of 1 mM (e.g. 50 µl from 1 M stock) and EGTA to final concentration of 0.1 mM (e.g. 10 µl from 0.5 M stock).
  4. HBSS stock with ethidium bromide
    For 5 µM ethidium bromide final concentration use 2.82 µl at 0.7 mg/ml ethidium bromide stock solution in per ml HBSS
  5. Acid washed coverslips
    Separate coverslips using tweezers and transfer them into a glass beaker containing an excess of a 1 M HCl solution. Make sure that the coverslips don’t stick to each other. Heat the coverslips to 50-60 °C for 4-16 h and carefully agitate from time to time. Discard the HCl solution and wash the coverslips extensively with distilled water (at least twice) followed by double-distilled water. Wash coverslips once with ethanol and store in ethanol at RT in a sealed container until use (e.g. a sterile 50 ml Falcon tube). Let the ethanol dry off each coverslip before you use it.

Acknowledgments

A.P. was a recipient of subsequent EMBO Long-Term and Marie Curie Intra-European Fellowships. This project was supported by ANR grant 2010 MIDI 007 01 to A.P. and P.J.S. and ERC Advanced Grant HOMEOEPITH to P.J.S. This protocol was adapted from “Retamal, M.A., Schalper, K.A., Shoji, K.F., Bennett, M.V., and Saez, J.C. (2007). Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential. Proc. Natl Acad. Sci. USA 104, 8322-8327” and a first short version of the adapted protocol was published in Puhar et al. (2013).

References

  1. Evans, W. H., De Vuyst, E. and Leybaert, L. (2006). The gap junction cellular internet: connexin hemichannels enter the signalling limelight. Biochem J 397(1): 1-14.
  2. Paemeleire, K., Martin, P. E., Coleman, S. L., Fogarty, K. E., Carrington, W. A., Leybaert, L., Tuft, R. A., Evans, W. H. and Sanderson, M. J. (2000). Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26. Mol Biol Cell 11(5): 1815-1827.
  3. Puhar, A. and Sansonetti, P. J. (2014). Induction of connexin-hemichannel opening. Bio-protocol 4(17): e1220.
  4. Puhar, A., Tronchère, H., Payrastre, B., Tran Van Nhieu, G. and Sansonetti, P. J. (2013). A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P. Immunity 39(6): 1121-1131.
  5. Tran Van Nhieu, G., Clair, C., Bruzzone, R., Mesnil, M., Sansonetti, P. and Combettes, L. (2003). Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat Cell Biol 5(8): 720-726.

简介

连接蛋白(Cxs)是脊椎动物的内在膜蛋白,其结合形成六聚体跨膜通道,称为半通道。已经描述了21种Cx类型,其根据它们的分子量命名。 Cxs在许多细胞类型中表达,例如上皮细胞,星形胶质细胞和免疫细胞。半通道允许高达1-2kDa的分子沿浓度梯度通过。当表面暴露时,半通道介导细胞溶质和细胞外空间之间的分子交换。半通道被默认关闭,但是已经描述了诱导其开放的几个线索,例如,细胞外Ca 2+浓度的下降(​​Evans等人, 2006);或用肠道病原体感染(Puhar et al。,2013; Tran Van Nhieu等人,2003)。半通道开放可以通过电生理学,通​​过定量半通道可渗透分子到胞外培养基中的释放或通过量化半通道可透性,质膜不渗透分子的吸收来测量。由于分子的摄取程度与其浓度,暴露时间,温度(这些参数是受控的)成比例的,并且重要的是与细胞表面上的活性半通道的数目成比例,摄取测定常规用于评估半通道开放。用于摄取荧光染料溴化乙锭的方案与用Cx26或Cx43稳定转染的Hela细胞一起使用(Paemeleire等人,2000)。然而,它可能可用于其他Cx表达细胞类型。

材料和试剂

  1. 感兴趣的细胞系和各种细胞培养基和从选择供应商传播的塑料制品
  2. DMEM低葡萄糖(Life Technologies,Gibco ,目录号:A1515401)
  3. 100x MEM非必需氨基酸(Life Technologies,Gibco ,目录号:11140050)
  4. 100×10,000U/ml青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15140122)
  5. 胎牛血清(FBS)(热灭活)(Life Technologies,Gibco ,目录号:10500056)
  6. Hank's缓冲盐溶液(HBSS)(无酚红)(Life Technologies,Gibco ,目录号:14025092)
  7. Hank's缓冲盐溶液(HBSS)(无钙,无镁,无酚红)(Life Technologies,Gibco ,目录号:14175095)
  8. 10x HBSS储备溶液而不是即用型缓冲液(Life Technologies,Gibco ,目录号:14065056和14185052)。
  9. 1 M HEPES(Life Technologies,Gibco ,目录号:15630080)
  10. 1 M MgCl 2
  11. 0.5 M Na-EGTA
  12. 溴化乙锭溶液(这里:eurobio,0.7mg/ml)
  13. 用于HelaCx26或HelaCx43细胞繁殖的培养基(参见配方)
  14. HBSS解决方案(参见配方)
  15. HBSS溶液缺乏Ca 2 + (参见配方)
  16. 含有溴化乙锭的HBSS储备液(见配方)
  17. 酸洗盖玻片(见配方)

设备

  1. 细胞培养孵育器
  2. 盖玻片和用于活细胞成像的支架或玻璃底皿(例如 MatTek)
  3. 水浴
  4. 移液器
  5. 愿望
  6. 清洁长椅
  7. 配有相机的荧光显微镜(理想的温度控制)

软件

  1. 图像分析软件[例如 ImageJ( http://imagej.nih.gov/ij/)或MetaMorph(Molecular Devices)]

程序

  1. 细胞在潮湿环境(培养箱)中在37℃和10%CO 2下生长,并根据标准方案传代。
  2. 在实验前一天将细胞接种在具有所需尺寸(例如24孔板)(参见Recipes)或玻璃 - 底盘的酸洗盖玻片上,并在70%汇合时使用。
  3. 制备不含含有溴化乙锭的Ca 2+的HBSS储备溶液(例如<5μM),以确保所有样品暴露于完全相同浓度的染料。
  4. 将细胞用温热(37℃)HBSS小心洗涤两次,并用不含Ca 2+的温热HBSS洗涤一次。对于阴性对照,仅使用HBSS。要清洗,小心吸出培养基或缓冲液,更换新鲜的缓冲液,小心吸取上下一次或两次,并更换新鲜的缓冲液。
  5. 在37℃下,用预热的5μM溴化乙锭在不含Ca 2+的HBSS中(以诱导半通道开放)加载细胞10分钟。对于阴性对照,使用预热的5μM溴化乙锭在含有Ca 2+ 2 +的HBSS中。
  6. 弃去上清液,用含有Ca 2+的温热HBSS洗涤细胞以诱导半通道闭合,从而将染料捕获在细胞溶质中。
  7. 在37℃和40x放大率下在30分钟内获得图像(溴化乙锭信号在此期间是稳定的)。确保采集样品中足够多的独立区域,并准备足够的独立重复进行定量。有关荧光显微镜的设置,请参阅注意事项。
  8. 细胞内溴化乙锭 - 使用图像分析软件定量单细胞的荧光。每个场减去背景荧光。

代表数据

  1. 代表结果


    图1.在HelaCx26细胞上进行的测定的典型结果。通过荧光定量摄取溴化乙锭,并将数值标准化为阳性对照(其中半通道开口由低细胞外Ca 2+诱导) > 2 + ),其被设置为100.在阴性对照中,没有诱导半通道开放,因此,背景荧光主要对应于通过内吞作用吸收的溴化乙锭。来自三个独立实验的具有约900个细胞的SD的平均值显示了18α-甘草次酸(AGA),100μM(半通道抑制剂)。

    图2.装有溴化乙锭的HelaCx26细胞的典型外观将细胞暴露于不含Ca 2+的HBSS中的溴化乙锭10分钟以诱导半通道开放(这种类型的样品对应于图1中的阳性对照)。显示了细胞的荧光A和透射B图像。为了定量,应当省略死亡的细胞和碎片(由B中的红色矩形突出显示)。垂死细胞的受损质膜通透性将允许溴化乙锭的非半通道依赖性传代。当染料与DNA结合时,溴化乙锭荧光增强
  2. 关于结果的可重复性和可变性的说明
    变化性低(10%),因为染料摄取与细胞群非常均匀。在阳性和阴性对照之间存在约两到三倍的差异。较短的溴化乙锭脉冲导致较高的变异性(由于处理时间的差异),而且阳性和阴性对照之间的差异略微较高,因为较少的溴化乙锭被内吞作用。

笔记

  1. 另请参阅Puhar和Sansonetti描述的协议"连接蛋白半通道开放诱导"(2014年)。
  2. 许多细胞系已丧失Cxs的表达,因此不具有半通道。确保感兴趣的细胞系有。
  3. 在实验过程中,小心处理细胞,因为半通道是机械敏感的。过度摇晃和剧烈的介质交换将引起半通道打开,因此增加了后续测量的背景
  4. 对于HBSS,可以使用10x储备溶液。
  5. 小心处理溴化乙锭,因为它是一种DNA插入剂,并在专用容器中处理含溴化乙锭的溶液。
  6. 对于单细胞实验,从显微镜图像的量化是强制性的。然而,如果需要批量测量,可以使用荧光板读取器代替显微镜
  7. 如果使用更高浓度的溴化乙锭,则应该减少孵育时间。然而,在非常短的孵育时间,处理时间的差异将使样品的比较更困难
  8. 溴化乙锭的激发和发射最大值分别为524nm和605nm。 因此,对于溴化乙锭,应使用绿色激发滤光片和橙色发射滤光片。 通常,可以使用Cy3/TRITC /罗丹明的标准显微镜设置,但为了获得最佳性能,可用过滤器的最佳组合应由实验者确定。

食谱

  1. 用于繁殖HelaCx26或HelaCx43细胞的培养基
    将500ml无菌DMEM与无菌5ml青霉素+链霉素(100U/ml青霉素和100μg/ml链霉素最终,任选)和50ml无菌FBS混合, 储存在4°C
  2. HBSS解决方案
    将HBSS与1M HEPES混合至终浓度为20mM(例如,1ml 1M HEPES,最终体积为50ml)
  3. HBSS溶液缺乏Ca 2 +
    将不含钙和镁的HBSS与1M HEPES混合至终浓度为20mM(例如,对于50ml终体积,例如1ml 1M HEPES),将MgCl 2终浓度(例如来自1μM储备液的50μl)和EGTA至终浓度0.1mM(例如来自0.5M储备液的10μl)。
  4. 含有溴化乙锭的HBSS库存
    对于5μM溴化乙锭的终浓度,使用2.82μl的0.7mg/ml溴化乙锭储备液/ml HBSS。
  5. 酸洗盖片
    使用镊子分开的盖玻片,并将其转移到含有过量的1M HCl溶液的玻璃烧杯中。确保盖玻片彼此不粘连。将盖玻片加热至50-60°C,4-16小时,并经常小心搅拌。丢弃HCl溶液,并用蒸馏水(至少两次),然后用双蒸水大量洗涤盖玻片。用乙醇洗涤盖玻片一次,并在室温下在密封容器中储存在乙醇中,直到使用(例如,无菌的50ml Falcon管)。让乙醇干燥每个盖玻片之前,使用它

致谢

A.P.是随后的EMBO长期和Marie Curie欧洲内部奖学金的接受者。该项目得到ANR拨款2010 MIDI 007 01至A.P.和P.J.S.和ERC高级授予HOMEOEPITH该方案改编自"Retamal,M.A.,Schalper,K.A.,Shoji,K.F.,Bennett,M.V。,和Saez,J.C。(2007)。连接蛋白43半通道的开放通过降低细胞内氧化还原电位而增加。 Proc。 Natl Acad。 Sci。 USA 104,8322-8327",并且适应的方案的第一简短版本在Puhar等人(2013)中公开。

参考文献

  1. Evans,W.H.,De Vuyst,E.and Leybaert,L。(2006)。 间隙连接细胞互联网:连接蛋白半通道进入信号传导。生化J 397(1):1-14。
  2. Paemeleire,K.,Martin,P.E.,Coleman,S.L.,Fogarty,K.E.,Carrington,W.A.,Leybaert,L.,Tuft,R.A.,Evans,W.H.and Sanderson,M.J。(2000)。 表达GFP-标记的连接蛋白43,32或26的HeLa细胞中的细胞间钙波。 Mol Biol Cell 11(5):1815-1827。
  3. Puhar,A。和Sansonetti,P.J。(2014)。 感应连接蛋白半通道开放 生物协议 4(17) :e1220。
  4. Puhar,A.,Tronchère,H.,Payrastre,B.,Tran Van Nhieu,G.and Sansonetti,P.J。(2013)。 Shigella 效应通过调节上皮释放的危险信号ATP来抑制炎症 生产脂质介体PtdIns5P。免疫 39(6):1121-1131。
  5. Tran Van Nhieu,G.,Clair,C.,Bruzzone,R.,Mesnil,M.,Sansonetti,P.and Combettes,L。(2003)。 连接蛋白依赖性细胞间通讯增加了志贺氏菌的侵袭和传播 epithelial cells。 Nat Cell Biol 5(8):720-726。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
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.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。