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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   

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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 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

  1. 6 cm tissue culture treated culture dishes (several distributers available e.g., Corning, Tewksbury, MA)
  2. GB 100-F8P borosilicate glass capillaries (Science Products GmbH, Hofheim, Germany)
  3. Syringe (1 ml)
  4. Spinal needle (0.5 mm, 25 G)
  5. Reaction tube
  6. Cell line or primary cell preparation of interest
  7. pcDNATM3.1/Zeo(+)
  8. HistoGreen HRP-subtrate Kit (Linaris, catalog number: E109 )
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  11. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  12. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  13. Neurobiotin (Vector Labaratories, catalog number: SP-1120 )
  14. Rhodamine B isothiocyanate-Dextran (Sigma-Aldrich, catalog number: R9379 )
  15. Tris base (Sigma-Aldrich, catalog number: T1503 )
  16. Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: L9650 )
  17. 50% glutaraldehyde solution (Sigma-Aldrich, catalog number: 340855 )
  18. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  19. Avidin D coupled horseradish peroxidase (Vector Labaratories, catalog number: A-2004 )
  20. PBS (see Recipes)
  21. Neurobiotin/Rhodamine B isothiocyanate-Dextran solution (see Recipes)
  22. LiCl solution (see Recipes)
  23. 0.5% glutaradehyde solution (see Recipes)
  24. Triton X-100 solution (see Recipes)
  25. Avidin D coupled horseradish peroxidase solution (see Recipes)

Equipment

  1. Microelectrode-holder suitable for 1 mm glass capillaries with AgCl electrode (e.g., World Precision Instruments, model: MEH8 )
  2. Dual Microiontophoresis current generator SYS-260 (World Precision Instruments, FL)
  3. Zeiss IM35 inverted fluorescence microscope (Zeiss, model: Zeiss IM35 ) equipped with:
    1. Heated stage set to 37 °C
    2. HBO lamp (100 W)
    3. Appropriate filter set to detect Rhodamine B fluorescence (Ex/Em 570/590; Filter Set 20 HE)
    4. The microscope should be placed on an anti-vibration microscope desk
  4. Incubator
  5. Micropipette puller P97 (Sutter Instrument, model: P-97 )
  6. Micromanipulator Injectman (Eppendorf, Hamburg, Germany)
  7. AgCl reference electrode (disc)

Procedure

  1. Setup
    Connect the micropipette holder to the positive and the reference electrode to the negative output of the microiontophoresis current generator. The microelectrode holder is mounted to the micromanipulator (Figure 1).


    Figure 1. Experimental setup. We use a Zeiss IM35 inverted fluorescence microscope equipped with heating stage (set to 37 °C), HBO fluorescence lamp (100 W) and appropriate filter set to detect Rhodamine B fluorescence. The microscope is placed on an anti-vibration table. The microelectrode holder is mounted to the micromanipulator and connected to the positive output of the microiontophoresis current generator. The reference electrode is connected to the negative output of the current generator (reference electrode and current generator are not shown on this photograph). 1: heating stage, 2: rod with connection for micropipette holder, 3: positive wire (iontophoresis current generator -> micropipette) 4: micromanipulator (moving in x-, y- and z-axis), 5: controller (micromanipulator), 6: temperature controller (heating stage), 7: power supply (fluorescence lamp), 8: power supply (micromanipulator), 9: power supply (microscope lamp), 10: anti-vibration microscope desk.

  2. Neurobiotin tracer microinjection
    1. Culture the cells on 6 cm cell culture dishes until they reach the desired confluency e.g., for HeLa cells we use 70-80% confluency.
      Note: Neurobiotin microinjection can be performed with any adherent cell line or primary cell culture to determine the extent of gap junctional intercellular communication.
    2. One hour prior to injection: Remove cell culture medium and wash the cells with PBS in order to remove cell debris. Aspirate the PBS and add 2 ml of fresh cell culture medium. Put the cells back into the incubator.
    3. Use the micropipette puller to manufacture injection capillaries with a tip size of 1-2 µm from GB 100-F8P borosilicate glass capillaries.
    4. Load the capillaries with Neurobiotin/Rhodamine B isothiocyanate-Dextran solution by bringing the backside of the micropipette in contact with the solution. The capillarity of the pipette will draw the fluid to the tip (Figure 2A).
    5. Fill the capillary with LiCl solution with a syringe by inserting a spinal needle into the backside of the capillary while withdrawing the needle as the micropipette fills. Avoid any air bubbles (Figure 2B). Little air bubbles can be removed by holding the micropipette tip down and gently flicking the pipette with the finger.
    6. Fill the micropipette holder with LiCl solution using syringe and needle. Avoid any air bubbles (Figure 2C).
    7. Carefully place the pipette into the micropipette holder. Avoid any air bubbles (Figure 2D).


      Figure 2. Loading the micropipette. A. Place the micropipette tip up into a 500 µl reaction tube containing 20 µl Neurobiotin/Rhodamine B isothiocyanate-Dextran solution. The capillarity of the pipette will draw the fluid to the tip. B. Fill the capillary with LiCl solution with a syringe by inserting a spinal needle into the backside of the capillary; C. Fill the micropipette holder with LiCl solution; D. Place the pipette into the micropipette holder. Avoid any air bubbles during all steps. Parts of this schematic were adapted from Servier Medical Art.

    8. Put the 6 cm culture dish containing the cells under the microscope (heated stage set to 37 °C) and place the reference electrode into the culture medium. Set up a negative retaining current to prevent tracer leakage from the micropipette.
      Note: Before starting microinjection, use a marker to draw a line at the bottom of the culture dish and place injections along this line. This facilitates going back to the injected cell under the microscope after staining.
    9. While looking at the cells through the microscope at 10x magnification, use the micromanipulator to carefully position the tip of the micropipette next to the cell you want to inject. Penetrate the cell with the pipette. Be careful not to break the pipette tip at the bottom of the culture dish. This procedure needs some practice.
    10. Switch from brightfield to fluorescence microscopy. The pipette tip should get visible due to red Rhodamin B fluorescence (E: 590 nm).
      Note: We recommend to work in a darkened room and keep main lights switched off during the experiment. This facilitates switching between brightfield and fluorescence microscopy.
    11. Inject the tracer solution by applying a positive current of 20 nA for 10-15 sec. The injected cell should fill with the red fluorescent dye. The dye should not spread into neighboring cells as Rhodamine B isothiocyanate-Dextran is too big to pass gap junction channels (Figure 3A, Inset).
    12. Carefully retract the pipette tip from the cell.
    13. Repeat the procedure to inject 10-15 cells per 6 cm dish within max. 15 min.

  3. Neurobiotin tracer visualisation
    1. Wash the injected cells twice with PBS directly after the last injection.
    2. Fix the cells with glutaraldehyde solution for 5 min at room temperature.
    3. Wash three times with PBS.
    4. Permeabilize cells over night at 4 °C using Triton X-100 solution.
    5. Wash cells three times with PBS.
    6. Incubate cells for 90 min at room temperature with Avidin D coupled horseradish peroxidase solution.
    7. Wash three times with PBS.
    8. Use the HistoGreen HRP-substrate Kit to indirectly visualize Neurobiotin. The use of HistoGreen-substrate will label cells which received the Neurobiotin tracer in blue (green) color (Figures 3A-3C).
      Notes:
      1. Alternatively to the HistoGreen HRP-substrate kit, 3,3’-Diaminobenzidine (DAB) staining can be used for visualization.
      2. Monitor the staining reaction by looking at the cells under the microscope to avoid overstaining. If the staining reaction should get too intense even after short incubation times dilute the Avidin D coupled horse radish peroxidase solution (Recipe 6) or reduce the amount of injected Neurobiotin by shortening the injection time (step B11).
    9. The number of coupled cells can be determined by manual counting of labeled cells.

Data analysis

For data analysis, we take photographs of every successfully injected cell using phase contrast microscopy (Figures 3A-3C). Some injections may fail due to breakage of the microcapillary tip or due to overstaining and are excluded from the analysis. We exclude an injection from analysis if the staining is so intense that single cells can no longer be discriminated from each other. The extent of gap junctional intercellular communication (GJIC) can be assessed by manual counting of labeled cells. We suggest to inject at minimum three individual 6 cm plates (try to perform 10-15 injections per plate) and count at least 8-10 injections on each plate to get a representative result. Usually we set the average number of coupled cells in the control sample (e.g., cells that have not been treated with pharmacological gap junction inhibitors) to 100% coupling and compare the other results to this (Figure 3D). As an example, representative data from two different experiments are presented:

  1. HeLa cells transfected with an expression plasmid (pcDNATM3.1/Zeo(+)) carrying the Cx43 ORF were injected with Neurobiotin. The tracer (blue color) was visualised using the HistoGreen HRP-substrate Kit. The white asterisk marks the injected cell. A spread of the tracer into neighboring cells can be observed (Figure 3A). The inset shows the fluorescence of Rhodamine B isothiocyanate-Dextran which was co-injected with Neurobiotin. The tip of the micropipette as well as the injected cell are visible (Figure 3A inset). The HeLa cell line used for these experiment does not show endogenous connexin expression. Therefore, a spread of the tracer is not observed when mock transfected HeLa cells are injected with Neurobiotin (Figure 3B).
  2. HM-1 mouse embryonic stem cells (HM-1 mESCs, Magin et al., 1992) were injected with Neurobiotin. mESCs endogenously express the gap junction proteins Cx31, Cx43 and Cx45 (Wörsdörfer et al., 2008 and 2016) (Figure 3C). Pre-incubation of mESCs with the pharmacological gap junction inhibitor 18-alpha glycyrrethinic acid concentration dependently (0-100 µm) decreased the number of coupled cells as assessed by Neurobiotin microinjection (Figure 3D).


    Figure 3. Microinjection of Neurobiotin. A. HeLa cells transfected with Cx43 were injected with Neurobiotin. The white asterisk marks the injected cell. The inset shows the fluorescence of Rhodamine B isothiocyanate-Dextran. B. The parental HeLa cell line used for this experiment does not show gap junctional intercellular communication; C. Mouse embryonic stem cells (mESCs) injected with Neurobiotin; D. Pre-incubation of mESCs with the gap junction inhibitor 18-alpha glycyrrethinic acid concentration dependently (0-100 µm) decreased the number of coupled cells. The number of coupling mESCs in the absence of the gap junction inhibitor was defined as 100%. Scale bars = 50 µm.

Notes

  1. In order to confirm the specificity of the assay the following control experiments should be performed:
    1. Monitor Neurobiotin microinjection by looking at the Rhodamine B fluorescence. The co-injected Rhodamine B isothiocyanate–Dextran is too big to pass gap junction channels and should not spread into neighboring cells.
    2. Cells can be incubated with pharmacological gap junction inhibitors (e.g., 18-alpha glycyrrethinic acid or its derivate carbenoxolone, Sigma-Aldrich, St. Louis, MO) prior to Neurobiotin microinjection. This should concentration dependently decrease the spread of dye into neighboring cells (Davidson et al., 1988; Figure 3D). Be aware that not all connexins are sensitive to these gap junction blockers: for example, Cx43 is sensitive to 18-alpha glycyrrethinic, while Cx31 and Cx45 are not (He et al., 2005; Wörsdörfer et al., 2008).

Recipes

  1. PBS
    133 mM NaCl
    2.7 mM KCl
    8.1 mM Na2HPO4
    1.5 mM KH2PO4
    in (dd)H2O
    pH 7.2
  2. Neurobiotin/Rhodamine B isothiocyanate-Dextran solution (20 µl per experiment)
    6% (w/v) Neurobiotin
    0.4% (w/v) Rhodamine B isothiocyanate-Dextran
    0.1 M Tris base
    in (dd)H2O
    pH 7.6
    Store at 4 °C, protect from light
  3. LiCl solution
    Note: Prepare 5-10 ml per experiment.
    1 M LiCl in (dd)H2O
  4. 0.5% glutaradehyde solution
    1% (v/v) 50% glutaraldehyde solution in PBS (final concentration 0.5%)
  5. Triton X-100 solution
    2% Triton X-100 in PBS
  6. Avidin D coupled horseradish peroxidase solution
    0.1% in PBS
    Prepare freshly before the experiment

Acknowledgments

Work in Bonn laboratory was supported by the German Research Foundation SFB645 project B2 and the Bonn Forum in Biomedicine to K.W. We thank Katharina Günther for proofreading the manuscript. Parts of the schematic procedure shown in Figure 2 were produced using the image bank of Servier Medical Art (www.servier.com) licensed under a Creative Commons Attribution 3.0 Unported License. The injection of Neurobiotin as a tracer for gap junctional communication between cultured cells has been often used in the literature. An excellent, extensive review on the permeability of connexin channels has been published by Harris and Locke (2009).

References

  1. Davidson, J. S. and Baumgarten, I. M. (1988). Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships. J Pharmacol Exp Ther 246(3): 1104-1107.
  2. Hampson, E. C., Vaney, D. I. and Weiler, R. (1992). Dopaminergic modulation of gap junction permeability between amacrine cells in mammalian retina. J Neurosci 12(12): 4911-4922.
  3. Harris, A. L. and Locke, D. (2009). Permeability of connexin channels. In: Harris and Locke (Eds.). Connexins: A guide. Humana Press.
  4. He, L. Q., Cai, F., Liu, Y., Liu, M. J., Tan, Z. P., Pan, Q., Fang, F. Y., Liang, D. S., Wu, L. Q., Long, Z. G., Dai, H. P., Xia, K., Xia, J. H. and Zhang, Z. H. (2005). Cx31 is assembled and trafficked to cell surface by ER-Golgi pathway and degraded by proteasomal or lysosomal pathways. Cell Res 15(6): 455-464.
  5. Kita, H. and Armstrong, W. (1991). A biotin-containing compound N-(2-aminoethyl)biotinamide for intracellular labeling and neuronal tracing studies: comparison with biocytin. J Neurosci Methods 37(2): 141-150.
  6. Magin, T. M., McWhir, J. and Melton, D. W. (1992). A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res 20(14): 3795-3796.
  7. Söhl, G. and Willecke, K. (2004). Gap junctions and the connexin protein family. Cardiovasc Res 62(2): 228-232.
  8. Wörsdörfer, P., Bosen, F., Gebhardt, M., Russ, N., Zimmermann, K., Komla Kessie, D., Sekaran, T., Egert, A., Ergün, S., Schorle, H., Pfeifer, A., Edenhofer, F. and Willecke, K. (2016). Abrogation of gap junctional communication in ES cells results in a disruption of primitive endoderm formation in embryoid bodies. Stem Cells 35(4): 859-871.
  9. Wörsdörfer, P., Maxeiner, S., Markopoulos, C., Kirfel, G., Wulf, V., Auth, T., Urschel, S., von Maltzahn, J. and Willecke, K. (2008). Connexin expression and functional analysis of gap junctional communication in mouse embryonic stem cells. Stem Cells 26(2): 431-439.

简介

可以通过将低分子量示踪物质显微注射到培养细胞中来评估相邻细胞之间的功能间隙连接通道。通过这种方法可以精确量化直接细胞间通信的程度。该方案描述了离子电渗注入和神经生物素在培养细胞中的可视化。

背景 间隙连接是在相邻细胞之间形成的细胞间通道,允许扩散交换低分子量分子(<1.8kDa)。间隙连接通道由两个彼此对接的半通道(连接器)组成。每个连接蛋白是称为连接蛋白(Cx)的蛋白质亚基的六聚体组装体。间隙连接蛋白基因家族由20只小鼠组成,21只在人体中组成。连接蛋白以其近似的kDa分子量命名,例如,Cx43具有43kDa的近似分子量(参见Söhl和Willecke,2004)。不同的连接蛋白在整个发育过程中广泛地表达在各种组织中,它们介导电和代谢偶联。此外,可以通过间隙连接通道直接扩散来交换第二信使分子和离子。
&NBSP; &NBSP;神经生物素(N-(2-氨基乙基)生物素)是在生理条件下为286Da分子量的化合物和+1的化合物。由于其小的尺寸,这个示踪剂甚至通过这些间隙连接通道,这些间隙连接通道对于较高分子量的其他常见示踪剂(例如,Lucifer Yellow或羧基荧光素)(Hampson等人, >,1992),因此代表检测间隙连接细胞间通讯的一种非常敏感的方法。与类似的示踪剂生物胞素相比,神经生物素似乎在溶解度和稳定性方面优越。此外,化合物可以选择性地以正电流离子电流并随后使用多聚甲醛或戊二醛固定(Kita和Armstrong,1991)。由于神经生物素不显示自身荧光,因此需要使用与辣根过氧化物酶缀合或直接连接荧光染料的抗生物素蛋白检测。

关键字:间隙连接, 连接蛋白, 神经生物素, 显微注射,  离子电渗疗法

材料和试剂

  1. 6厘米组织培养处理的培养皿(几种分配器,例如,可康奈治,马萨诸塞州,Tewksbury)
  2. GB 100-F8P硼硅玻璃毛细管(Science Products GmbH,Hofheim,Germany)
  3. 注射器(1 ml)
  4. 脊髓针(0.5mm,25G)
  5. 反应管
  6. 感兴趣的细胞系或原代细胞制备
  7. pcDNA TM / / sup> 3.1 / Zeo(+)
  8. HistoGreen HRP-subtrate试剂盒(Linaris,目录号:E109)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  10. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  11. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  12. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  13. 神经生物素(Vector Labaratories,目录号:SP-1120)
  14. 罗丹明B异硫氰酸酯 - 葡聚糖(Sigma-Aldrich,目录号:R9379)
  15. Tris碱(Sigma-Aldrich,目录号:T1503)
  16. 氯化锂(LiCl)(Sigma-Aldrich,目录号:L9650)
  17. 50%戊二醛溶液(Sigma-Aldrich,目录号:340855)
  18. Triton X-100(Sigma-Aldrich,目录号:X100)
  19. 抗生物素蛋白D偶联辣根过氧化物酶(Vector Labaratories,目录号:A-2004)
  20. PBS(见配方)
  21. 神经生物素/罗丹明B异硫氰酸酯 - 葡聚糖溶液(参见食谱)
  22. LiCl溶液(参见食谱)
  23. 0.5%戊二醛溶液(见配方)
  24. Triton X-100溶液(参见食谱)
  25. 抗生物素蛋白D偶联辣根过氧化物酶溶液(参见食谱)

设备

  1. 适用于具有AgCl电极的1mm玻璃毛细管的微电极支架(例如,World Precision Instruments,型号:MEH8)
  2. 双微电泳电流发生器SYS-260(世界精密仪器,FL)
  3. 蔡司IM35倒置荧光显微镜(Zeiss,型号:Zeiss IM35)配备:
    1. 加热阶段设置为37°C
    2. HBO灯(100W)
    3. 适当的过滤器组,以检测罗丹明B荧光(Ex / Em 570/590;过滤器组20 HE)
    4. 显微镜应放置在抗振显微镜台上
  4. 孵化器
  5. 微型拔片机P97(Sutter Instrument,型号:P-97)
  6. Micromanipulator注射器(Eppendorf,Hamburg,Germany)
  7. AgCl参考电极(光盘)

程序

  1. 设置
    将微量移液器支架连接到正极和参比电极到微离子电渗电流发生器的负极。微电极支架安装在显微操纵器上(图1)

    图1.实验设置我们使用配有加热阶段(设定为37℃),HBO荧光灯(100W)和适当的过滤器组的Zeiss IM35倒置荧光显微镜来检测罗丹明B荧光。显微镜放置在防振台上。微电极保持器安装在显微操纵器上,并与微电泳电流发生器的正极输出相连。参考电极连接到电流发生器的负输出(参考电极和电流发生器未在本照片上显示)。 1:加热台,2:连接用于微量移液器支架的杆,3:正极线(离子电渗发生器 - >微量吸管)4:显微操纵器(在x轴,y轴和z轴上移动),5:控制器(显微操纵器) ,6:温度控制器(加热台),7:电源(荧光灯),8:电源(显微操作器),9:电源(显微镜灯),10:防振显微镜台。
  2. 神经生物素示踪剂显微注射
    1. 培养6cm细胞培养皿上的细胞直至达到希望的融合,例如,对于HeLa细胞,我们使用70-80%融合。
      注意:可以用任何贴壁细胞系或原代细胞培养物进行神经生物素显微注射,以确定间隙连接细胞间通讯的程度。
    2. 注射前1小时:取出细胞培养液,用PBS清洗细胞,以除去细胞碎片。吸入PBS并加入2ml新鲜细胞培养基。将细胞放回孵化器。
    3. 使用微量移液器拉杆从GB 100-F8P硼硅玻璃毛细管制造尖端尺寸为1-2μm的注射毛细管。
    4. 通过使微量吸液管的背面与溶液接触,将神经生物素/多巴胺B异硫氰酸酯 - 葡聚糖溶液装载毛细管。移液管的毛细管力会将流体吸入尖端(图2A)
    5. 当毛细管充满时,将针头插入毛细管的背面,同时用针注射器将LiCl溶液填充到LiCl溶液中。避免任何气泡(图2B)。可以通过将微量移液器吸头向下移动并用手指轻轻移动移液管来移除小气泡
    6. 使用注射器和针头将微量移液管支架装入LiCl溶液。避免任何气泡(图2C)。
    7. 小心地将移液器放入微量移液器支架。避免任何气泡(图2D)。


      图2.装载微量移液管。A.将微量吸头顶部放入含有20μl神经生物素/异硫氰酸罗丹明B - 葡聚糖溶液的500μl反应管中。移液器的毛细作用将吸引流体到尖端。 B.通过将脊髓针插入毛细管的背面,用注射器向毛细管填充LiCl溶液; C.向微量移液器支架上填充LiCl溶液; D.将移液器放入微量移液器支架。在所有步骤中避免任何气泡。该示意图的一部分改编自Servier Medical Art。

    8. 将含有细胞的6cm培养皿置于显微镜下(加热阶段设定为37℃),并将参比电极置于培养基中。设置负的保持电流,以防止示踪剂从微量移液器泄漏 注意:在开始显微注射之前,使用标记在培养皿底部绘制一条线,并沿着该线注入。这有助于在染色后在显微镜下回到注射的细胞。
    9. 通过显微镜以10倍放大倍率观察细胞时,请使用显微操纵器小心地将微量移液管的尖端放置在要注射的细胞旁边。用移液管渗透细胞。小心不要打破培养皿底部的移液器尖端。此过程需要一些练习。
    10. 从明场切换到荧光显微镜。由于红色罗丹明B荧光(E:590 nm),移液管尖应该可见。
      注意:我们建议在黑暗的房间内工作,并在实验过程中关闭主灯。这有助于在明场和荧光显微镜之间切换。
    11. 通过施加20 nA的正电流10-15秒来注入示踪剂溶液。注射细胞应填充红色荧光染料。染料不应扩散到相邻的细胞中,因为若丹明B异硫氰酸酯 - 葡聚糖太大而不能通过间隙连接通道(图3A,Inset)。
    12. 小心地从细胞中收集移液器尖端。
    13. 重复这个步骤,每6 cm最多注入10-15个细胞。 15分钟。

  3. 神经生物素示踪剂可视化
    1. 用戊二醛溶液在室温下固定细胞5分钟
    2. 用PBS洗三次。
    3. 使用Triton X-100溶液在4℃下使细胞过夜
    4. 用PBS洗涤细胞三次。
    5. 用Avidin D偶联辣根过氧化物酶溶液在室温孵育细胞90分钟
    6. 用PBS洗三次。
    7. 使用HistoGreen HRP底物试剂盒间接显现神经生物素。使用HistoGreen-底物将以蓝色(绿色)颜色标记接受神经生物素示踪剂的细胞(图3A-3C)。
      注意:
      1. 除了HistoGreen HRP-底物试剂盒之外,可以使用3,3'-二氨基联苯胺(DAB)染色观察。
      2. 通过在显微镜下观察细胞来监测染色反应,以避免过度染色。如果即使在短时间内染色反应也应该变得太强烈,稀释抗生物素蛋白D偶联的辣根过氧化物酶溶液(配方6)或通过缩短注射时间来减少注射的神经生物素的量(步骤B11)。 >
    8. 可以通过手动计数标记细胞来确定偶联细胞的数量

数据分析

对于数据分析,我们使用相差显微镜拍摄每个成功注射的细胞(图3A-3C)。一些注射可能由于微毛细血管尖端的破裂或由于过度染色而失效,并被排除在分析之外。如果染色非常强烈,我们可以排除注射液,使单细胞不能再被区分开来。间隙连接细胞间通讯(GJIC)的程度可以通过手动计数标记细胞进行评估。我们建议注射至少三个单独的6孔板(尝试每个板进行10-15次注射),并计数至少8-10次注射,以获得代表性的结果。通常我们将对照样品(例如,尚未用药理学间隙连接抑制剂处理的细胞)中的偶联细胞的平均数设置为100%偶联,并将其他结果与此比较(图3D) 。作为一个例子,提出了两个不同实验的代表性数据:

  1. 用携带Cx43 ORF的表达质粒(pcDNA 3.1 / Zeo(+))转染的HeLa细胞注射神经生物素。使用HistoGreen HRP-底物试剂盒可视化示踪剂(蓝色)。白色星号标记注入的细胞。可以观察到示踪剂扩散到相邻的细胞中(图3A)。插图显示了与神经生物素共注射的罗丹明B异硫氰酸酯 - 葡聚糖的荧光。微量吸头的尖端以及注入的细胞是可见的(图3A插图)。用于这些实验的HeLa细胞系不显示内源性连接蛋白表达。因此,当用神经生物素注射模拟转染的HeLa细胞时,不观察到示踪剂的扩散(图3B)。
  2. 用神经生物素注射HM-1小鼠胚胎干细胞(HM-1 mESC,Magin等人,1992)。 mESCs内源性表达间隙连接蛋白Cx31,Cx43和Cx45(Wörsdörfer等人,2008和2016)(图3C)。通过神经生物素显微注射评估,mESCs与药理学间隙连接抑制剂18-α甘氨酸浓度的依赖性(0-100μm)浓度的预孵育减少了偶联细胞的数量(图3D)。


    图3.显微注射神经生物素 A.用Cx43转染的HeLa细胞注射神经生物素。白色星号标记注入的细胞。插图显示了罗丹明B异硫氰酸酯 - 葡聚糖的荧光。 B.用于本实验的亲本HeLa细胞系不显示间隙连接的细胞间通讯; C.用神经生物素注射的小鼠胚胎干细胞(mESCs) D.间接连接抑制剂18-α甘氨酸浓度依赖性(0-100μm)的mESCs的预孵育减少了偶联细胞的数量。在没有间隙连接抑制剂的情况下,偶联mESCs的数目定义为100%。刻度棒=50μm。

笔记

  1. 为了确认测定的特异性,应进行以下对照实验:
    1. 通过观察罗丹明B荧光监测神经生物素显微注射。共注射的罗丹明B异硫氰酸酯 - 葡聚糖太大,不能通过间隙连接通道,不应扩散到相邻的细胞中。
    2. 在神经生物素显微注射之前,可将细胞与药理学间隙连接抑制剂(例如,18-α甘氨酸或其衍生物碳烯醇,Sigma-Aldrich,St.Louis,MO)一起温育。这应该浓度依赖性地降低染料在相邻细胞中的扩散(Davidson等人,1988;图3D)。请注意,并不是所有的连接蛋白对这些间隙连接阻断剂都敏感:例如,Cx43对18-α甘草次苷敏感,而Cx31和Cx45不是(He&et al。,2005;Wörsdörfer > et al。,2008)。

食谱

  1. PBS
    133 mM NaCl
    2.7 mM KCl
    8.1mM Na 2 HPO 4
    1.5mM KH PO 4
    在(dd)H 2 O O
    中 pH 7.2
  2. 神经生物素/罗丹明B异硫氰酸酯 - 葡聚糖溶液(每个实验20μl)
    6%(w / v)神经生物素
    0.4%(w / v)若丹明B异硫氰酸酯 - 葡聚糖
    0.1 M Tris碱
    在(dd)H 2 O O
    中 pH 7.6
    储存于4°C,避光保护
  3. LiCl溶液
    注意:每个实验准备5-10毫升。
    (dd)H 2 O中的1M LiCl
  4. 0.5%戊二醛溶液
    1%(v / v)50%戊二醛溶液PBS(终浓度0.5%)
  5. Triton X-100解决方案
    2%Triton X-100在PBS中
  6. 抗生物素蛋白D偶联辣根过氧化物酶溶液
    PBS中0.1%,
    在实验前新鲜准备

致谢

波恩实验室的工作由德国研究基金会SFB645项目B2和生物医学博鳌论坛向K.W.提供支持。我们感谢KatharinaGünther对手稿进行校对。使用Servier Medical Art的图像库( www.servier.com )根据Creative Commons Attribution 3.0 Unported License许可。在文献中经常使用注射神经生物素作为培养细胞之间间隙连接连通的示踪剂。 Harris和Locke(2009)发表了一篇关于连接蛋白通道渗透性的广泛综述。

参考

  1. Davidson,JS和Baumgarten,IM(1988)。&nbsp; Glycyrrhetinic酸衍生物:一类新颖的间隙连接细胞间通讯抑制剂。结构活性关系。药物治疗药物 246(3):1104-1107。
  2. Hampson,EC,Vaney,DI and Weiler,R。(1992)。&nbsp; 哺乳动物视网膜中无长突细胞之间的间隙连接通透性的多巴胺能调节。 J Neurosci 12(12):4911-4922。
  3. Harris,AL和Locke,D。(2009)。&nbsp; 连接信道的透过率。在哈里斯和洛克(Eds。)。连接蛋白:指南。人文出版社。
  4. 他,LQ,Cai,F.,Liu,Y.,Liu,MJ,Tan,ZP,Pan,Q.,Fang,FY,Liang,DS,Wu,LQ,Long,ZG,Dai,HP,Xia,K 。,Xia,JH和Zhang,ZH(2005)。&nbsp; Cx31通过ER-高尔基体途经组装并投递到细胞表面,并被蛋白酶体或溶酶体途径降解。 15(6):455-464。
  5. Kita,H.和Armstrong,W.(1991)。一种含有生物素的化合物N-(2-氨基乙基)生物素酰胺,用于细胞内标记和神经元追踪研究:与生物体素的比较.J Neurosci Methods 37(2):141-150。 br />
  6. Magin,TM,McWhir,J.和Melton,DW(1992)。一种具有良好的细菌系贡献和基因靶向频率的新型小鼠胚胎干细胞系。 核酸研究20(14):3795-3796。
  7. Söhl,G.和Willecke,K.(2004)。间隙连接点和连接蛋白家族。 Cardiovasc Res 62(2):228-232。
  8. Wörsdörfer,P.,Bosen,F.,Gebhardt,M.,Russ,N.,Zimmermann,K.,Komla Kessie,D.,Sekaran,T.,Egert,A.,Ergün,S.,Schorle, ,Pfeifer,A.,Edenhofer,F.和Willecke,K.(2016)。 ES细胞中间隙连接通讯的消除导致胚状体内原始内胚层形成的破坏。 35(4):859-871。 br />
  9. Wörsdörfer,P.,Maxeiner,S.,Markopoulos,C.,Kirfel,G.,Wulf,V.,Auth,T.,Urschel,S.,von Maltzahn,J.and Willecke,K。(2008) ; 连接蛋白在小鼠胚胎干细胞中间隙连接通讯的表达和功能分析细胞。 干细胞 26(2):431-439。
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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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