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Transepithelial Electrical Resistance (TER) measurement is a reliable and efficient method to quantify the permeability of barrier forming cells such as epithelial cells. Measuring the permeability of the epithelial cells will help the researchers to investigate the barrier function of epithelium in various infectious and inflammatory diseases. Here we provide a real-time and impedance-based approach for measuring the permeability of epithelial cell monolayer using the Electrical Cell Substrate Impedance Sensing (ECIS®) instrumentation.

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Study of Epithelium Barrier Functions by Real-time TER Measurement
实时TER测量法研究上皮组织的屏障功能

免疫学 > 粘膜免疫学 > 上皮
作者: Jingjing Meng
Jingjing MengAffiliation: Department of Surgery, University of Minnesota Medical School, Minneapolis, USA
Bio-protocol author page: a3146
 and Sabita Roy
Sabita RoyAffiliation: Department of Surgery, University of Minnesota Medical School, Minneapolis, USA
For correspondence: royxx002@umn.edu
Bio-protocol author page: a3147
Vol 6, Iss 10, 5/20/2016, 1347 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1815

[Abstract] Transepithelial Electrical Resistance (TER) measurement is a reliable and efficient method to quantify the permeability of barrier forming cells such as epithelial cells. Measuring the permeability of the epithelial cells will help the researchers to investigate the barrier function of epithelium in various infectious and inflammatory diseases. Here we provide a real-time and impedance-based approach for measuring the permeability of epithelial cell monolayer using the Electrical Cell Substrate Impedance Sensing (ECIS®) instrumentation.
Keywords: ECIS(企业竞争情报系统), Barrire(barrire), Epithelium(上皮)

[Abstract]

Materials and Reagents

  1. Epithelial cell lines (e.g. Caco-2, IEC-6, CMT-93 cell lines)
  2. DMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
  3. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  4. Collagen (Type I, Rat tail) (Corning, catalog number: 354236 )
  5. L-cysteine (Sigma-Aldrich, catalog number: C7352 )
  6. Lipopolysaccharide from E. coli 0111:B4 (Sigma-Aldrich, catalog number: L4391 )
  7. Saline (Sigma-Aldrich, catalog number: S8776 )
    Note: It is also named as “Sodium chloride solution (0.9%)” on Sigma-Aldrich website.
  8. Acetic acid (Sigma-Aldrich, catalog number: A6283 )
  9. Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  10. Collagen solution (see Recipes)
  11. Cysteine solution (see Recipes)
  12. Cell culture medium (see Recipes)

Equipment

  1. ECIS Zθ (Theta) and 16 W array station (Applied BioPhysics, http://www.biophysics.com/products-ecisz0.php)
  2. 8W10E+ Cultureware (Figure 1)


    Figure 1. 8W10E+ Cultureware (Applied BioPhysics, http://www.biophysics.com/cultureware.php#link8)

Software

  1. ECIS Zθ Software

Procedure

  1. Clean and coat the cultureware
    Note: Two methods are available for cleaning the electrodes in the 8W10E+ cultureware (cysteine treatment or electrical stabilization).

    Cysteine treatment
    1. Place 200 µl of 10 mM sterile cysteine solution in each well for 10 min. To rinse the wells, put 500 µl of sterile medium, saline or water and aspirate the liquid. Repeat the rinse step twice.
    2. Place 300 µl, 35 µg/ml collagen solution in each well and incubate at room temperature for one hour. To rinse the wells, put 500 µl of sterile medium, saline or water and aspirate the liquid. Repeat the rinse step twice.

    Electrical stabilization
    1. Add 300 µl, 35 µg/ml collagen solution in each well and incubate at room temperature for one hour. To rinse the wells, put 500 µl of sterile medium, saline or water and aspirate the liquid. Repeat the rinse step twice.
    2. Add 200 µl medium to each well.
    3. Put the cultureware in the ECIS Array station and run electrical stabilization as follows:
      1. Place the cultureware in the ECIS Array station within the incubator (Figure 2). Start ECIS Zθ Software.
      2. Click the “Set up” button to check if arrays are properly connected to the system.
      3. If the array diagram in the lower left hand position of the screen turns green, the array is properly connected. Red color indicates the absence of an array or an incorrectly connected array. If the array diagram shows red, adjust the position of the cultureware slightly and click “check” to ascertain that each well of the cultureware is connected properly.
      4. Click the “Stabilize” button (Figure 3). Eletrical stabilization should only be used on cell-free electrods.
    4. After stabilization, remove the medium.


      Figure 2. Cultureware in the ECIS array station within the incubator

  2. Inoculate epithelial cells in the cultureware
    1. Prepare single cell suspension and add 400 µl of cell suspension into each well. The number of cells inoculated in each well can range from 104 to 105, which will depend on the characteristic of cells. For the intestinal epithelial cells, 5 x 104 are usually plated in each well.
    2. After plating cells in the wells, wait 20 to 30 min before placing the array in the incubator space.

  3. Acquiring data
    1. Put the cultureware containing cells in the ECIS Array station and run setup procedure (1) to check that all wells are properly connected (2) as shown in Figure 3.


      Figure 3. ECIS Software and set-up procedure

    2. Select “Multiple frequency” (4) and click start (5) as shown in Figure 3. The system will start to record the impedance at 11 pre-defined frequencies. Click “R” (6) and the resistance values will show up over time during the experiment. High frequency is usually used to monitor whether the cells form the confluent monolayer. While the cells are spreading, the resistance values are increasing at 4,000 Hz as shown in Figure 4. When the cells reach confluence, the resistance curve will reach the plateau, at this point the treatment can be added to test the change of barrier function.


      Figure 4. The attachment and spreading behaviors of IEC-6 cell. 5 x 104 of IEC-6 cells were inoculated in the ECIS cultureware. The cells formed the confluent monolayer after 60 hand at this time point the curve reached the plateau.

    3. Due to the sensitivity of the instrument to the media components, it is preferable to change media every day during the entire duration of the experiment. Click Pause and take out the cultureware. Withdraw the old medium and gently add fresh medium everyday. When the cells reach confluence, withdraw 200 µl medium and gently add 200 µl of the 2x stimulus-containing medium so that the final centration is 1x. Lipopolysaccharide (LPS) with the final concentration of 1 μg/ml can be used as a positive control.
    4. At the desired time point, click Finish (7) to stop the experiment and analyze the data.
    5. Low frequency (500 Hz or 250 Hz) is usually used to monitor the barrier function of the cell monolayer. Choose the desired time point as time 0 and select “Normalize” (Figure 5). According to the product manual, for tight epithelial cells, impedance at low frequencies is more sensitive and provides a very effective measure of the layer's barrier function. The normalized resistance curve at 500 Hz is shown in Figure 6. The results indicate that LPS disrupted the barrier function of IEC-6 cells.


      Figure 5. Data normalization


      Figure 6. Disruption of barrier function following LPS stimulation. Saline or 1 μg/ml LPS were added to the IEC-6 monolayer. The reduced resistance values indicate the disrupted barrier function. Green dash line indcted the time point when LPS was added.

    6. If you need to calculate the average resistance values for multiple wells. Usually, each treatment was run in triplicates and the average resistance values for each treatment group can be compared using statistical analysis. Select the desired wells (2) and select “group” (8) (Figure 3). Then click . The average resistance curve with error bars will show up automatically.

Notes

  1. While cleaning and coating the cultureware, avoid using Phosphate-buffered saline (PBS) since PBS has been shown to interfere with the absorption of some proteins by the metal electrode. Cysteine is highly recommended to obtain consistent results. But if cysteine interacts with protein coatings on the electrodes, electrical stabilization is preferred.
  2. For IEC-6 cells, collagen solution was used to coat the cultureware. Other proteins such as gelatin and fibronectin can also be used according to the characteristics of different cells.
  3. The impedance recording is very sensitive to temperature change. Before taking out the cultureware from the incubator, warm up the media at 37 °C firstly.
  4. If the cultureware is not connected properly, no data will be collected. Therefore, click “check” to confirm the proper connection each time when putting the cultureware in the station.

Recipes

  1. Collagen solution
    35 µg/ml collagen
    17.5 mM acetic acid solution was prepared by diluting acetic acid with sterile distilled water (1:1,000 dilution). The collagen is dissolved in 17.5 mM acetic acid solution.
  2. Cysteine solution
    10 mM cysteine
    Sterile distilled water
  3. Cell culture medium
    DMEM
    10% FBS
    1% Penicillin/Streptomycin

Acknowledgements

The protocol had been adapted from a previously published paper (Meng et al., 2015) and the Ztheta Manual provided by the Applied Biophysics, Inc. This work was supported in part by the NIH grants RO1 DA 12104, RO1 DA 022935, RO1 DA031202, K05DA033881, P50 DA 011806 and 1R01DA034582 to S. Roy.

References

  1. Meng, J., Banerjee, S., Li, D., Sindberg, G. M., Wang, F., Ma, J. and Roy, S. (2015). Opioid exacerbation of gram-positive sepsis, induced by gut microbial modulation, is rescued by IL-17A neutralization. Sci Rep 5: 10918.
  2. ECIS Operation Manual Version 1.2.123: http://www.biophysics.com/publications/ECIS_Operation_Manual_v1_2_123.pdf.

材料和试剂

  1. 上皮细胞系(例如Caco-2,IEC-6,CMT-93细胞系)
  2. DMEM培养基(Thermo Fisher Scientific,Gibco TM ,目录号:11965-092)
  3. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10082147)
  4. 胶原(I型,大鼠尾)(Corning,目录号:354236)
  5. L-半胱氨酸(Sigma-Aldrich,目录号:C7352)
  6. 来自大肠杆菌0111:B4(Sigma-Aldrich,目录号:L4391)的脂多糖
  7. 盐水(Sigma-Aldrich,目录号:S8776)
    注意:在Sigma-Aldrich网站上也将其命名为"氯化钠溶液(0.9%)"。
  8. 乙酸(Sigma-Aldrich,目录号:A6283)
  9. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  10. 胶原蛋白溶液(见配方)
  11. 半胱氨酸溶液(参见配方)
  12. 细胞培养基(参见配方)

设备

  1. ECISZθ(Theta)和16W阵列站(Applied BioPhysics, http://www.biophysics.com /products-ecisz0.php
  2. 8W10E +文化软件(图1)


    图1. 8W10E +文化软件(Applied BioPhysics, http://www.biophysics .com/cultureware.php#link8

软件

  1. ECISZθ软件

程序

  1. 清洁并涂抹培养皿
    注意:有两种方法可用于清洁8W10E +培养皿中的电极(半胱氨酸处理或电稳定)。

    半胱氨酸治疗
    1. 放置200微升10毫米无菌的半胱氨酸溶液在每个孔10分钟。要冲洗孔,放500μl无菌培养基,盐水或水和 吸出液体。 重复冲洗步骤两次。
    2. 放置300微升, 35μg/ml胶原溶液,并在室温下孵育   一小时。 要冲洗孔,放500μl无菌培养基,盐水 或水并且抽吸液体。 重复冲洗步骤两次。

    电稳定
    1. 在每个孔中加入300μl,35μg/ml胶原溶液,并在室温下孵育   温度下1小时。 要冲洗孔,放500μl无菌 介质,盐水或水,并抽吸液体。 重复冲洗步骤 两次。
    2. 每孔加入200μl培养基。
    3. 将培养皿放在ECIS Array站中,按照以下步骤运行电气稳定:
      1. 将培养皿放在培养箱内的ECIS Array站中(图2)。 启动ECISZθ软件。
      2. 单击"设置"按钮,检查阵列是否正确连接到系统。
      3. 如果数组图在屏幕的左下角位置 变为绿色,阵列已正确连接。 红色表示 不存在数组或不正确连接的数组。 如果数组 图表显示红色,稍微调整文化餐具的位置 单击"检查"以确定培养皿的每个孔 连接正确。
      4. 单击"稳定"按钮(图3)。 电稳定只应用于无细胞电极。
    4. 稳定后,取出介质。


      图2.孵化器内的ECIS阵列工作站中的文化工具

  2. 接种培养皿中的上皮细胞
    1. 准备单细胞悬液,并添加400微升的细胞悬浮液 每个井。 在每个孔中接种的细胞的数目可以是 10 4 至10 5 ,这将取决于电池的特性。 为了 肠上皮细胞,5×10 8个通常在每个孔中铺板
    2. 在孔中铺平细胞后,等待20至30分钟,然后将阵列放入培养箱空间。

  3. 获取数据
    1. 将包含单元格的文化工具放入ECIS Array工作站并运行 设置程序(1)检查所有油井是否正确连接(2) 如图3所示。


      图3. ECIS软件和设置过程

    2. 选择"多频率"(4)并单击开始(5),如图所示 图3.系统将开始记录11处的阻抗 预定义频率。点击"R"(6)和电阻值将 在实验期间随时间显示。通常使用高频 以监测细胞是否形成汇合单层。虽然 细胞扩散,电阻值在4,000Hz时增加  如图4所示。当细胞达到汇合时,电阻 曲线将达到平台,此时可以加入处理 以测试屏障功能的变化

      图4.附件和  IEC-6细胞的扩散行为。 5 x 10 4 个IEC-6细胞 接种在ECIS培养皿中。细胞形成汇合单层后60手在此时点曲线达到 高原。

    3. 由于仪器的灵敏度 媒体组件,最好每天更换媒体 整个实验持续时间。 点击暂停并取出 文化。 取出旧培养基,轻轻加入新鲜培养基 每天。 当细胞达到汇合,撤回200微升培养基和 轻轻加入200微升含2x刺激的培养基,使最终   中心为1x。 脂多糖(LPS)与终浓度   的1μg/ml可用作阳性对照
    4. 在所需的时间点,单击完成(7)停止实验并分析数据
    5. 低频(500 Hz或250 Hz)通常用于监视屏障功能的细胞单层。选择所需的时间点  时间0并选择"Normalize"(图5)。根据产品 手动,对于紧张的上皮细胞,阻抗在低频下更多  并提供对层的屏障的非常有效的测量 功能。 500Hz下的归一化电阻曲线如图所示 结果表明LPS破坏了IEC-6的屏障功能  细胞

      图5.数据标准化


      图6.中断 LPS刺激后的屏障功能。 盐水或1μg/ml LPS 添加到IEC-6单层。减小的电阻值表示 破坏屏障功能。绿色虚线表示时间点 当添加LPS时
    6. 如果你需要计算平均值 多个孔的电阻值。 通常,每次处理都进行   一式三份和每个处理组的平均电阻值 可以使用统计分析进行比较。 选择所需的孔(2)   并选择"group"(8)(图3)。 然后点击。 平均电阻   带有误差棒的曲线将自动显示。

笔记

  1. 在清洁和涂覆培养皿时,避免使用磷酸盐缓冲盐水(PBS),因为PBS已显示干扰一些蛋白质被金属电极吸收。 强烈建议使用半胱氨酸以获得一致的结果。 但是如果半胱氨酸与电极上的蛋白质涂层相互作用,则优选电稳定
  2. 对于IEC-6细胞,使用胶原溶液涂覆培养皿。 根据不同细胞的特性,也可以使用其它蛋白质如明胶和纤连蛋白
  3. 阻抗记录对温度变化非常敏感。 在从培养箱中取出培养皿之前,首先在37℃下预热培养基。
  4. 如果文化工具未正确连接,则不会收集数据。 因此,每次将文化用品放入工作站时,单击"检查"以确认正确的连接

食谱

  1. 胶原蛋白溶液
    35μg/ml胶原蛋白 通过用无菌蒸馏水(1:1000稀释)稀释乙酸来制备17.5mM乙酸溶液。 将胶原溶解在17.5mM乙酸溶液中
  2. 半胱氨酸溶液
    10mM半胱氨酸 无菌蒸馏水
  3. 细胞培养基
    DMEM
    10%FBS
    1%青霉素/链霉素

致谢

该方案改编自之前发表的论文(Meng等人,2015)和Applied Biophysics,Inc。提供的Ztheta手册。该工作部分由NIH授予RO1 DA 12104 ,RO1 DA 022935,RO1 DA031202,K05DA033881,P50 DA 011806和1R01DA034582。

参考文献

  1. Meng,J.,Banerjee,S.,Li,D.,Sindberg,G. M.,Wang,F.,Ma,J.and Roy,S。 由肠道微生物调节诱导的革兰氏阳性脓毒症的阿片样物质恶化,被IL-17A中和 。 Sci Rep 5:10918.
  2. ECIS操作手册1.2.123版: http://www.biophysics.com/publications/ECIS_Operation_Manual_v1_2_123.pdf 。
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How to cite this protocol: Meng, J. and Roy, S. (2016). Study of Epithelium Barrier Functions by Real-time TER Measurement. Bio-protocol 6(10): e1815. DOI: 10.21769/BioProtoc.1815; Full Text



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