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Induction of Connexin-hemichannel Opening
Connexin八次跨膜通道白的诱导打开   

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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

  1. Cell line of interest and assorted cell culture media and plasticware for propagation from supplier of choice
  2. 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)]
  3. Salmonella enterica serovar Typhimurium (for example wild-type strain SL1344)
  4. Enteropathogenic Escherichia coli (EPEC) (for example serotype O127: H6 wild-type strain E2348/69)
  5. Biocoat intestinal epithelium differentiation environment (BD Biosciences, catalog number depends on well size)
  6. DMEM low glucose (Life Technologies, Gibco®, catalog number: A1515401 )
  7. 100x MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140050 )
  8. 100x 10,000 U/ml penicillin-streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
  9. Fetal bovine serum (FBS) (heat-inactivated) (Life Technologies, Gibco®, catalog number: 10500056 )
  10. Hank’s buffered salt solution (HBSS) (no phenol red) (Life Technologies, Gibco®, catalog number: 14025092 )
  11. Hank’s buffered salt solution (HBSS) (no calcium, no magnesium, no phenol red) (Life Technologies, Gibco®, catalog number: 14175095 )
  12. 10x HBSS stock solutions instead of ready to use HBSS if desired (Life Technologies, Gibco®, catalog numbers: 14065056 and 14185052 )
  13. 1 M HEPES (Life Technologies, Gibco®, catalog number: 15630080 )
  14. 1 M MgCl2
  15. 0.5 M Na-EGTA
  16. 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]
  17. Growth media for bacteria containing Congo red (see Recipes)
  18. Culture medium for propagation of TC7 cells (see Recipes)
  19. Culture medium for propagation of HelaCx26 or HelaCx43 cells (see Recipes)
  20. HBSS solution (see Recipes)
  21. HBSS solution devoid of Ca2+ (see Recipes)

Equipment

  1. Incubator for cell culture
  2. Cover slips of desired size (e.g. 24 well plates) (if desired)
  3. Water bath
  4. Pipettes
  5. Aspiration
  6. Clean bench
  7. Incubator for bacterial cultures
  8. Centrifuge adapted to spin culture plates (for infections with EPEC)

Procedure

  1. Cells were kept in matching medium at 37 °C and 10% CO2 in a humid environment (incubator) and passaged according to standard protocols.
  2. Cells were seeded in culture plates or on cover slips of desired size (e.g. 24 well plates) the day before the experiment and used at 70-80% confluency. For infections, cells were kept in medium devoid of antibiotics for at least 24 h.
  3. To obtain polarized TC7 cells the Biocoat Intestinal Epithelium Differentiation Environment was used according to the protocol provided by the producer.

    To induce hemichannel opening with low extracellular Ca2+
    1. Cells were carefully washed twice with warm (37 °C) HBSS and once with warm HBSS devoid of Ca2+. For polarized TC7 cells both the apical and the basolateral sides have to be washed with buffer (by carefully lifting the transwell chambers containing the cells with tweezers and substituting the buffer in the well). For negative controls, only HBSS was used.
    2. Fresh warm HBSS devoid of Ca2+ was added and cells were incubated at 37 °C (e.g. in an incubator, a heated microscope or floating in a water bath). For polarized TC7 cells both the apical and the basolateral sides have exposed to HBSS devoid of Ca2+. For negative controls, HBSS was used.
    3. Hemichannels can be closed again by exchanging HBSS devoid of Ca2+ with HBSS.

    To induce hemichannel opening with Shigella
    1. Cells were carefully washed twice with HBSS at RT just before infection. Cells are washed and kept at RT (rather than using warm buffer) to synchronize infection. Indeed, the activity of the type III secretion system (T3SS-the main virulence determinant in Shigella, Salmonella and EPEC) is temperature-dependent, for example in Shigella the T3SS is not active below 35 °C. Therefore, samples are shifted to 37 °C to trigger infection only after bacterial adhesion to the cell surface.
      Bacterial strains were conserved as glycerol stocks at -80 °C. Shigella was streaked out on soybean-casein digest agar containing 0.01% (w/v) Congo red and grown over-night at 37 °C to select virulent colonies (virulent colonies are red, colonies that have lost the virulence plasmid are whitish). A single colony was picked, grown over-night at 37 °C and 200 rpm in soybean-casein digest broth, with antibiotics if indicated, and a 200-fold dilution sub-cultured to OD600 nm = 0.3-0.5.
    2. Bacteria were diluted in HBSS at RT, added to non-polarized cells at RT at a multiplicity of infection (MOI) of 1: 5 (target cell: bacteria) and allowed to attach for 15 min. For polarized TC7 cells, bacteria were added on the apical side at an MOI of 1: 40.
    3. HBSS was removed to eliminate non-adhered bacteria (in polarized cells the buffer has to be exchanged both on the apical and basolateral side to control the temperature), warm (37 °C) HBSS was added and cells were incubated at 37 °C (e.g. in an incubator or floating in a water bath) for the desired time (e.g. 15 min).

    To induce hemichannel opening with Salmonella
    1. Cells were carefully washed twice with HBSS at RT just before infection.
      Bacterial strains were conserved as glycerol stocks at -80 °C. Salmonella was streaked out on Luria-Bertani (LB)-agar to isolate colonies and grown overnight at 37 °C. The next evening a single colony was picked, grown overnight at 37 °C and 200 rpm in LB-broth, with antibiotics if indicated.
    2. Bacteria were diluted in HBSS at RT and added at RT (on the apical side for polarized TC7 cells) at a multiplicity of infection (MOI) of 1: 2.5 (target cell: bacteria).
    3. Cells were incubated at 37 °C (e.g. in an incubator or floating in a water bath) for the desired time (e.g. 2 min-infections with Salmonella proceed very quickly to bacterial adhesins).

    To induce hemichannel opening with EPEC
    1. Cells were carefully washed twice with HBSS at RT just before infection.
      Bacterial strains were conserved as glycerol stocks at -80 °C. EPEC was streaked out on Luria-Bertani (LB)-agar to isolate colonies and grown overnight at 37 °C. The next evening a single colony was picked, grown overnight at 37 °C and 200 rpm in LB-broth, with antibiotics if indicated.
    2. Bacteria were grown over night, diluted in HBSS at RT and added at RT (on the apical side for polarized TC7 cells) at a multiplicity of infection (MOI) of 1: 2.5 (target cell: bacteria).
    3. Bacteria were centrifuged onto cells at 180 x g for 10 min at RT by spinning the culture plate in an appropriate centrifuge to promote their adhesion.
    4. HBSS was removed to eliminate non-adhered bacteria (in polarized cells the buffer has to be exchanged both on the apical and basolateral side to control the temperature), warm (37 °C) HBSS was added and cells were incubated at 37 °C (e.g. in an incubator or floating in a water bath) for the desired time (e.g. 2 h-infections with EPEC proceed very slowly.

Representative data

  1. Notes about reproducibility and variability in results.
    1. If hemichannel opening is induced by lowering the extracellular Ca2+ concentration, variability is lower (10-20%) than when induced by infection (20-30%). It is best to prepare triplicates of quadruplicates for infections (ideally from separate cultures to avoid dilution-dependent artifacts).
    2. If hemichannels do not open upon exposure to medium devoid of Ca2+, residual Ca2+ might be present (e.g. because the washing steps were not performed carefully).

Notes

  1. Many cell lines have lost expression of Cxs and therefore do not have hemichannels. Make sure your cell line of interest does.
  2. 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.
  3. For HBSS, 10x stock solutions may be used.
  4. For infections, HBSS may be substituted with DMEM devoid of phenol red for washes and incubations with bacteria.
  5. If you wish to quantify molecules that are released when hemichannels open, use sufficient (cells should be well covered, e.g. 200 µl in a 24-well plate), but not high volumes of buffer during incubations, or else the molecules of interest will be unnecessarily diluted.
  6. Shigella, Salmonella and EPEC differ in their capacity to adhere to epithelial cells and the speed at which adhesion and infection take place. Therefore, the steps in which bacteria are added and the incubation times have to be adapted.

Recipes

  1. Culture medium containing Congo red
    Prepare a 1% (weight/volume) concentrated stock solution of Congo red (100x), for example 0.5 g Congo red powder in 50 ml distilled water. Sterilize by filtration using filters with 0.2 µm pore size and sufficient filtration capacity for the volume you prepare. Add Congo red stock solution to liquid Soybean-Casein Digest Agar, for example, after autoclaving the dissolved powder, when it has cooled to about 55 °C. Add Congo red to a final concentration of 0.01% (weight/volume), for example 5 ml Congo red stock solution in 500 ml growth medium.
  2. Culture medium for propagation of TC7 cells
    Mix 500 ml sterile DMEM with 5 ml sterile penicillin + streptomycin (100 U/ml penicillin and 100 µg/ml streptomycin final, optional), 5 ml sterile non-essential amino acids (from 100x stock solution) and 50 ml sterile FBS.
    Stored at 4 °C
  3. 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 sterile 50 ml FBS
    Stored at 4 °C
  4. 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)
  5. HBSS solution devoid of Ca2+
    Mix HBSS devoid of calcium and magnesium with 1M 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).

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. A first short version of this 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). Dye-uptake Experiment through Connexin Hemichannels. Bio-protocol 4(17): e1221.
  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)。该方案与上皮细胞,特别是与极化和非极化肠上皮TC7细胞和用Cx26或Cx43稳定转染的Hela细胞(Paemeleire等人,2000)一起使用。然而,它可能可用于其他Cx表达细胞类型。是否半通道是开放的可以通过电生理学或通过测量半通道可渗透分子(例如,ATP)在细胞外介质中的释放或半通道可渗透的质膜不渗透分子的摄取来确定[例如,荧光染料溴化乙锭 - 参见相关协议"通过Connexin Hemichannels进行染料吸收实验"(Puhar和Sansonetti,2014 )]。

关键字:半通道, 感染, 志贺菌, 沙门氏菌, 致病性大肠杆菌(EPEC)

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. 表达粘附素AfaE的[例如,血清型5a野生型菌株M90T,血清型5a非侵入性mxiD突变体(例如,血清型5a野生型菌株M90T,血清型5a非侵入性mxiD突变体 对于阴性对照)或血清型5a ipgD - 突变体(在用该菌株感染时,半通道将保持开放延长时间)]
  3. 沙门氏菌鼠伤寒沙门氏菌(例如野生型菌株SL1344)
  4. 致病性大肠杆菌(EPEC)(例如血清型O127:H6野生型菌株E2348/69)
  5. Biocoat肠上皮分化环境(BD Biosciences,目录号取决于孔大小)
  6. DMEM低葡萄糖(Life Technologies,Gibco ,目录号:A1515401)
  7. 100x MEM非必需氨基酸(Life Technologies,Gibco ,目录号:11140050)
  8. 100×10,000U/ml青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15140122)
  9. 胎牛血清(FBS)(热灭活)(Life Technologies,Gibco ,目录号:10500056)
  10. Hank's缓冲盐溶液(HBSS)(无酚红)(Life Technologies,Gibco ,目录号:14025092)
  11. Hank's缓冲盐溶液(HBSS)(无钙,无镁,无酚红)(Life Technologies,Gibco ,目录号:14175095)
  12. 10x HBSS储备溶液而不是即可使用HBSS(Life Technologies,Gibco ,目录号:14065056和14185052)。
  13. 1 M HEPES(Life Technologies,Gibco ,目录号:15630080)
  14. 1 M MgCl 2
  15. 0.5 M Na-EGTA
  16. 细菌生长培养基[例如Luria-Bertani肉汤(Life Technologies,目录号:10855001); 根据制造商的说明书(Life Technologies,目录号:22700025)针对沙门氏菌和EPEC制备的Luria-Bertani琼脂粉末; 胰蛋白酶大豆肉汤(大豆酪蛋白消化培养基,BD,目录号:257107); 根据制造商的说明书制备的胰蛋白酶大豆琼脂粉末(大豆 - 酪蛋白消化琼脂培养基,Difco,目录号:236950),用于志贺氏菌]
  17. 含有刚果红的细菌的生长培养基(参见配方)
  18. 用于繁殖TC7细胞的培养基(参见配方)
  19. 用于HelaCx26或HelaCx43细胞繁殖的培养基(参见配方)
  20. HBSS解决方案(参见配方)
  21. HBSS溶液缺乏Ca 2 + (参见配方)

设备

  1. 细胞培养孵育器
  2. 所需尺寸的盖玻片(例如 24孔板)(如果需要)
  3. 水浴
  4. 移液器
  5. 愿望
  6. 清洁长椅
  7. 细菌培养孵化器
  8. 离心机适于旋转培养板(用于EPEC感染)

程序

  1. 将细胞保持在37℃和10%CO 2的匹配培养基中,在潮湿环境(培养箱)中,并根据标准方案传代。
  2. 在实验前一天将细胞接种在培养板或所需大小的盖玻片(例如24孔板)上,并在70-80%融合度下使用。 对于感染,将细胞保持在没有抗生素的培养基中至少24小时
  3. 为了获得极化的TC7细胞,根据生产商提供的方案使用Biocoat肠上皮分化环境。

    为了诱导半通道开放与低细胞外Ca <
    1. 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。对于极化的TC7细胞,顶端和基底外侧都必须用缓冲液洗涤(通过用镊子小心地提起含有细胞的transwell腔室并替换孔中的缓冲液)。对于阴性对照,仅使用HBSS
    2. 加入不含Ca 2+的新鲜温热HBSS,并将细胞在37℃(例如在培养箱,加热的显微镜中或漂浮在水浴中)温育。对于极化的TC7细胞,顶端和基底外侧都暴露于缺乏Ca 2+的HBSS。对于阴性对照,使用HBSS
    3. 半通道可以通过交换没有Ca 2 + 的HBSS与HBSS再次闭合。

    使用志贺氏菌引起半通道开放
    1. 在感染之前,将细胞在室温下用HBSS仔细洗涤两次。洗涤细胞并保持在室温(而不是使用温热缓冲液)以同步感染。事实上,III型的活性 分泌系统(T3SS-志贺氏菌,沙门氏菌和EPEC中的主要毒力决定子)是温度依赖性的,例如在志贺氏菌中 T3SS不是活性低于35℃。因此,将样品转移到37℃以仅在细菌粘附到细胞表面之后触发感染 细菌菌株作为甘油储备在-80℃保存。 在含有0.01%(w/v)刚果红的大豆 - 酪蛋白消化琼脂上划线,并在37℃下生长过夜以选择毒性菌落(毒性菌落为红色,菌落丢失毒力质粒是白色的)。挑取单个菌落,在37℃和200rpm下在大豆 - 酪蛋白消化肉汤中生长过夜,如果指示,用抗生素培养,并将200倍稀释度培养至OD 600nm = 0.3-0.5。
    2. 将细菌在室温下在HBSS中稀释,在室温下以1:5的多重感染(MOI)(靶细胞:细菌)加入到非极化细胞中,并允许附着15分钟。对于极化的TC7细胞,在顶端侧以1:40的MOI添加细菌
    3. 去除HBSS以消除非粘附的细菌(在极化的细胞中,缓冲液必须在顶端和基底外侧交换以控制温度),加热(37℃)HBSS并将细胞在37℃( 例如 15分钟),在孵化器中或漂浮在水浴中)。例如

    用沙门氏菌诱导半通道开放
    1. 在感染之前,将细胞用HBSS在室温下仔细洗涤两次 细菌菌株作为甘油储备在-80℃保存。将沙门氏菌在Luria-Bertani(LB)琼脂上划线以分离菌落并在37℃下生长过夜。第二天晚上,挑选单个菌落,在37℃和200rpm的LB-肉汤中生长过夜,如果指示,用抗生素。
    2. 将细菌在室温下在HBSS中稀释,并在RT(对于极化的TC7细胞在顶侧上)以1:2.5的多重感染(MOI)(靶细胞:细菌)加入。
    3. 将细胞在37℃(例如在孵育器中漂浮或在水浴中漂浮)孵育所需时间(例如 2分钟 - 用沙门氏菌感染) em>很快进行细菌粘附素)。

    使用EPEC 启动半通道开启
    1. 在感染之前,将细胞用HBSS在室温下仔细洗涤两次 细菌菌株作为甘油储备在-80℃保存。将EPEC在Luria-Bertani(LB) - 琼脂上划线以分离集落,并在37℃下生长过夜。第二天晚上,挑选单个菌落,在37℃和200rpm的LB-肉汤中生长过夜,如果指示,用抗生素。
    2. 使细菌生长过夜,在室温下在HBSS中稀释并在RT(对于极化的TC7细胞在顶侧上)以1:2.5的多重感染(MOI)(靶细胞:细菌)加入。
    3. 通过在合适的离心机中旋转培养板以促进它们的粘附,将细菌在室温下以180×g离心10分钟。
    4. 去除HBSS以消除非粘附的细菌(在极化的细胞中,缓冲液必须在顶端和基底外侧交换以控制温度),加热(37℃)HBSS并将细胞在37℃( 例如在孵化器中或漂浮在水浴中)持续所需时间(例如 2小时 - 用EPEC进行的感染进行得很慢。

代表数据

  1. 注意结果的重现性和变异性。
    1. 如果通过降低细胞外Ca 2+浓度诱导半通道开放,则变异性比由感染诱导(20-30%)更低(10-20%)。 最好制备一式四份的感染(理想情况下从单独的文化,以避免稀释依赖的文物)。
    2. 如果半通道在暴露于缺乏Ca 2+ 2 +的培养基时不开放,则可能存在残留的Ca 2+ 2+(例如),因为洗涤步骤 不仔细执行)。

笔记

  1. 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。
  2. 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
  3. 对于HBSS,可以使用10x储备溶液
  4. 对于感染,HBSS可以用不含酚红的DMEM代替用于与细菌的洗涤和温育
  5. 如果希望定量在半通道开放时释放的分子,使用足够的(细胞应该被良好覆盖,例如在24孔板中200μl),但是在孵育期间不是高体积的缓冲液,否则所关注的分子将被不必要地稀释
  6. ,沙门氏菌和EPEC在粘附上皮细胞的能力和粘附和感染发生的速度方面不同。因此,添加细菌的步骤和孵育时间必须适应

食谱

  1. 含有刚果红的培养基
    准备刚果红(100x)的1%(重量/体积)浓缩储备溶液,例如在50ml蒸馏水中的0.5g刚果红粉末。使用0.2μm孔径的过滤器过滤灭菌,并为您准备的体积提供足够的过滤能力。向液体大豆 - 酪蛋白消化琼脂中加入刚果红贮备液,例如,当高压灭菌溶解的粉末,当其已经冷却至约55℃时。加入刚果红至终浓度为0.01%(重量/体积),例如在500ml生长培养基中的5ml刚果红贮备液。
  2. 用于繁殖TC7细胞的培养基
    将500ml无菌DMEM与5ml无菌青霉素+链霉素(100U/ml青霉素和100μg/ml链霉素最终,任选),5ml无菌非必需氨基酸(来自100x储备溶液)和50ml无菌FBS混合。 br /> 储存在4°C
  3. 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)和无菌50ml FBS混合
    储存在4°C
  4. 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)
  5. 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至终浓度 1mM(例如来自1M储备液的50μl)和EGTA至终浓度0.1mM(例如来自0.5M储备液的10μl)。

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 该协议的第一个简短版本发布在Puhar et al。(2013)。

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。 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)。 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)。 通过Connexin Hemichannels进行染料吸收实验 生物协议 4(17 ):e1221。
  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。 上皮细胞。 Nat Cell Biol 5(8):720-726
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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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