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Jun 2014

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Induction of Epithelial-mesenchymal Transition in MDCK II Cells
诱导MDCK II细胞上皮-间充质转化   

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Abstract

Epithelial-mesenchymal transition (EMT) is a reversible process of epithelial cell transdifferentiation into a mesenchymal cell, that enables initiation of cell migration. EMT plays an important role in embryonic development, tissue repair and cancer metastasis. Better understanding of cellular and molecular events during EMT will not only provide novel insights on how mammalian organism develops and how epithelial tissues regenerate, but also can identify novel therapeutic targets for cancer therapy. Here we aim to provide a detailed protocol on how to induce EMT in Madin-Darby Canine Kidney (MDCK) II epithelial cell line and perform immunofluorescent staining on EMT-induced cells.

Keywords: Epithelial-mesenchymal transition (上皮细胞间质转型), MDCK cells (MDCK细胞), Hepatocyte growth factor (肝细胞生长因子), Epithelial plasticity (上皮细胞可塑性), EMT (上皮细胞间质转型), Mesenchymal cell differentiation from epithelial cell (从上皮细胞分化为间充质细胞)

Background

Epithelial cells are characterized by cell plasticity, that is the ability to adopt different cellular phenotypes (Carter et al., 2019; Yuan et al., 2019). Epithelial-mesenchymal transition (EMT) is a form of epithelial cell plasticity. During EMT epithelial cells disrupt cell-cell junctions, lose their polarity and change their shape from squamous, cuboidal, or columnar into spindle-like and become migratory, thus gaining the properties of mesenchymal cells (Kalluri and Weinberg, 2009). EMT can be evaluated by immunostainings and measurement of expression levels of markers such as E-cadherin, ZO-1, vimentin, fibronectin and N-cadherin (Kalluri and Weinberg, 2009). The studies on EMT during the last decade centered mostly on the role of transcription factors e.g., Snail1/2, ZEB1/2, Twist and microRNAs (Gonzalez and Medici, 2014), cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (Yadav et al., 2011; Li et al., 2012), and signaling mechanisms such as transforming growth factor beta (TGF-β) signaling and NF-κB signaling (Gonzalez and Medici, 2014; Song et al., 2014; Dongre and Weinberg, 2019). However, regulatory mechanisms of early stages EMT and the precise sequence of molecular events during EMT remain largely unknown.


Madin-Darby Canine Kidney (MDCK) epithelial cell line is a cellular model to study epithelial cell polarity and junctions (Dukes et al., 2011; Yonemura, 2014; Vidal-Quadras et al., 2017). MDCK II cell line is a strain, that originates from higher passage of parental MDCK cells (Dukes et al., 2011). MDCK II cells differ from parental MDCK cells in that MDCK II cells have leaky cell junctions. Importantly, MDCK cells can be cultured in a standard 2D cell culture system, as well as in a more physiologically relevant 3D cell culture system (Yonemura, 2014; Vidal-Quadras et al., 2017).


EMT can be easily stimulated in 2D cell cultures of MDCK II cells by treatment with hepatocyte growth factor (HGF), that decreases cell roundness, upregulates the expression of vimentin, and increases the cell distance to the first and the second neighbor cell (Farrell et al., 2014). Here we provide a detailed description of the procedure on how to stimulate EMT in 2D cell cultures of MDCK II cells. Additionally, we describe the procedure for immunostaining of EMT-induced cells for ZO-1, a tight junction protein, and vimentin, the classical EMT marker.


Materials and Reagents

  1. Sterile Cell Culture Dish, 100 × 20 mm (Sarstedt, catalog number: 83.3902 )

  2. Serological pipettes: 5 ml (Sarstedt, catalog number: 86.1253.001 ), 10 ml (Sarstedt, catalog number: 86.1254.001 ), 50 ml (Sarstedt, catalog number: 86.1256.001 )

  3. Tube 15 ml (Sarstedt, catalog number: 62.554.502 )

  4. Tube 50 ml (Sarstedt, catalog number: 62.547.254 )

  5. 1.5 ml microtubes (Sarstedt, catalog number: 72.690.301 )

  6. Culture chambers: Lumox 8-well specimen slide detachable (Sarstedt, catalog number: 94.6150.801)

    Note: The procedure also works with FalconTM Chambered Cell Culture Slides, 8 wells, (Corning, catalog number: 354118).

  7. Laboratory bottle, borosilicate 3.3 glass, 50 ml (VWR, catalog number: 215-3261 )

  8. Laboratory bottle, borosilicate 3.3 glass, 1,000 ml (VWR, catalog number: 215-1595 )

  9. Aluminium foil

  10. ARTTM Barrier Specialty Pipette Tips: 10 µl (Thermo Scientific, catalog number: 2140 ), 200 µl (Thermo Scientific, catalog number: 2770 ), 1,000 µl (Thermo Scientific, catalog number: 2279 )

  11. Microscope slide holder

  12. Cover slips Menzel Glaser 24 × 60 mm (ThermoFisher Scientific, catalog number: E-4137 )

  13. Parafilm

  14. MDCK II cell line (Merck/Sigma-Aldrich, catalog number: ECACC 00062107 )

  15. Eagle's Minimum Essential Medium (EMEM), 500 ml (ATCC, catalog number: 30-2003 )

  16. Fetal bovine serum (FBS), 500 ml (Gibco, catalog number: 10270-106)

    Note: FBS does not require heat inactivation.

  17. Dulbecco's phosphate-buffered saline (DPBS) 1×, no calcium, no magnesium, 500 ml (Gibco, catalog number: 14190-094 )

  18. Trypsin-EDTA (0.25%), phenol red, 100 ml (Gibco, catalog number: 25200056 )

  19. Recombinant Human HGF (HEK293 derived), 25 μg (Peprotech, catalog number: 100-39H )

  20. Goat normal serum, 10 ml (Agrisera, catalog number: AS10 1548 )

  21. Saponin, 10 g (Sigma-Aldrich, catalog number: S4521 )

  22. Paraformaldehyde (PFA), 1 kg (Sigma-Aldrich, catalog number: 16005 )

  23. Sodium chloride (NaCl), 1 kg (VWR Chemicals, catalog number: 27810.295 )

  24. Potassium chloride (KCl), reagent grade, Reag. Ph Eur, 1 kg (Scharlau, catalog number: PO02001000 )

  25. di-Sodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), 1 kg (Scharlau, catalog number: SO03391000 )

  26. Potassium dihydrogen phosphate (KH2PO4), 1 kg (Merck, catalog number: 1048731000 )

  27. VECTASHIELD® Antifade Mounting Medium with DAPI, 10 ml (Vector Laboratories, catalog number: H-1200 )

  28. Nail polish

  29. Sodium hydroxide (NaOH), 1 kg (Merck, catalog number: 1064691000 )

  30. Primary antibodies (Table 1)


    Table 1. Primary antibodies


  31. Secondary antibodies (Table 2)


    Table 2. Secondary antibodies


  32. Complete medium for MDCK II cells (see Recipes)

  33. PBS 10× (see Recipes)

  34. PBS 1× (see Recipes)

  35. Blocking buffer (see Recipes)

  36. Washing buffer (see Recipes)

  37. 3% PFA (see Recipes)

  38. 5% saponin (see Recipes)

Equipment

  1. Counting chamber, type Burker (Hirschmann, catalog number: 8100201 )

  2. Fume hood

  3. Eppendorf® Research® plus pipette, 3-pack: 0.5-10 µl, 10-100 µl, 100-1,000 µl (Sigma-Aldrich, catalog number: Z683884 )

  4. Integra Pipetboy 2 (VWR, catalog number: 612-0927 )

  5. Heating and magnetic stirrer RH basic 2 (Dulova, catalog number: QSA337 )

  6. Centrifuge VWR Compact Star CS4

  7. 37 °C, 5% CO2 cell culture incubator

  8. Standard phase contrast microscope

  9. Confocal microscope Zeiss 710

Software

  1. ImageJ (https://imagej.nih.gov/ij/download.html)

Procedure

  1. Starting MDCK II cell culture

    1. Prepare the complete medium for MDCK II cells (see Recipes).

    2. Thaw a vial with frozen MDCK II cells.

    3. Transfer the cells into a 15 ml Falcon tube containing the complete medium.

    4. Centrifuge the cells for 5 min at 1,500 rpm (214 × g).

    5. Discard the supernatant.

    6. Add 10 ml of complete medium. Pipette up and down.

    7. Transfer the cells into a cell culture dish.

    8. Culture cells at 37 °C, 5% CO2 in cell culture incubator.


  2. MDCK II cell passage

    1. Pre-warm complete medium and trypsin.

    2. Remove medium from the cells.

    3. Wash with 10 ml of DPBS.

    4. Add 2 ml of trypsin.

    5. Incubate cells for 5 min at 37 °C.

    6. Check under the microscope if cells are detached.

      Note: If cells are not detached, continue incubation for 2-5 min at 37 °C.

    7. Add 10 ml of complete medium.

    8. Pipette up and down.

    9. Transfer the cell suspension into a 15 ml tube.

    10. Centrifuge the cells for 5 min at 1,500 rpm (214 × g).

    11. Discard the supernatant.

    12. Seed the cells in a new cell culture dish. Use split ratio of 1:5.

    13. Culture cells at 37 °C.


  3. Induction of EMT

    Day 0

    1. Seed 16,000 cells/well in an 8-well chamber slide.

      Note: For the induction of EMT in MDCK II cells, the cells were passaged less than 10 times.

    2. Incubate cells overnight.


    Day 1

    1. Prepare HGF stock 100 μg/ml and make aliquots:

      1. Spin down the vial with lyophilized HGF.

      2. Add 250 μl of sterile BSA 1 mg/ml (in PBS) to the vial with HGF.

      3. Mix by pipetting.

      4. Spin down.

      5. Make aliquots. Use sterile 1.5 ml tubes.

      6. Store the aliquots at -80 °C.

    2. Dilute the HGF stock with complete medium to final concentration 20 ng/ml. Mix by vortexing.

    3. Remove medium from the cells, that were seeded in the 8-well chamber slide.

    4. Add 0.5 ml of DPBS per well.

    5. Remove DPBS.

    6. Add complete medium containing HGF, use 0.5 ml medium per well.

      Note: For the control, treat cells with complete medium without HGF.

    7. Incubate the cells for 24 h at 37 °C.


  4. Immunofluorescent staining and imaging

    Day 2

    1. Thaw:

      3% PFA (see Recipes)

      Goat serum

      5% saponin (see Recipes)

    2. Prepare:

      Washing Buffer (see Recipes)

      Blocking Buffer (see Recipes)

    3. Remove culture medium.

    4. Fix the cultures in 3% PFA for 10-15 min. Use 0.5 ml of PFA/well.

      Note: All immunostaining steps are performed at room temperature, except where otherwise provided.

    5. Wash the cultures with PBS 1× (see Recipes). Use 0.5 ml of PBS 1×/well.

    6. Add 0.5 ml of Blocking Buffer/well. Incubate for 1 h at room temperature.

    7. Incubate the cultures with primary antibodies (Table 1), overnight, at 4 °C. Cover the cultures with parafilm.

      1. Dilute anti-ZO-1 antibody at a ratio 1:200.

      2. Dilute anti-vimentin antibody at a ratio 1:500.

      3. Use Washing Buffer for dilution of antibodies.

      4. Use minimum 200 μl antibody solution/well.


    Day 3

    1. Remove antibody solution.

    2. Wash with PBS 1×. Use 0.5 ml of PBS 1×/well.

    3. Incubate the cultures with fluorescent secondary antibodies (Table 2) for 1 h.

      1. Dilute secondary antibodies at a ratio 1:500.

      2. Use Washing Buffer for dilution of antibodies.

      3. Use minimum 200 μl antibody solution/well.

      4. Cover the cultures with aluminium foil to protect from the light.

    4. Wash with PBS 1×. Use 0.5 ml of PBS 1x/well.

    5. Discard PBS 1×.

    6. Remove media chamber. Use removing device.

    7. Mounting

      1. Put few drops of mounting medium on a coverslip slide.

      2. Put the coverslip slide on the top of the slide containing the cultures. Avoid bubbles.

    8. Seal the mounted slide with nail polish.

    9. Store the slides in a microscope slide folder at 4 °C. Protect from light.


    Day 4

    Take images of the stained cells with a confocal microscope (Figure 1).

Data analysis

Confocal microscope images are analyzed with ImageJ software. In addition to phenotypical characterization by immunofluorescence, EMT-induced MDCK cells can be analyzed by western blotting, evaluation of cell roundness as well as measurement of the cell distance to the first and the second neighbor cell, as described by Farrell et al. (2014).



Figure 1. Phenotypical characterization of EMT in MDCK II cells. A. Experimental schedule: MDCK II cells were treated with hepatocyte growth factor (HGF) for 24 h, fixed and then stained for EMT markers. B. Immunostaining for vimentin and tight junction protein-1 (zonula occludens-1, ZO-1). DAPI was used for nuclear counterstaining. Objective 20×. Scale bar, 50 μm.

Notes

For successful and reproducible results, we recommend:

  1. To strictly follow the manufacturer’s guidelines on HGF storage and to avoid repeated HGF freeze-thaw cycles.

  2. That MDCK II cells should be weekly observed under the microscope to monitor the stability of phenotype.

  3. That MDCK II cultures should never be overgrown (avoid > 80% confluency).

  4. That cell culture media should be exchanged regularly (avoid acidic medium, that can be identified by color change from pink into yellow, as it can induce EMT).

Recipes

  1. Complete medium for MDCK II cells

    450 ml Eagle's Minimum Essential Medium (EMEM)

    50 ml fetal bovine serum (FBS)

  2. PBS 10×

    80 g NaCl

    2.2 g KCl

    14.2 g Na2HPO4·2H2O

    2 g KH2PO4

    Adjust final volume to 1 L with distilled water

  3. PBS 1×

    100 ml PBS 10×

    900 ml distilled water

  4. Blocking buffer

    500 μl goat serum

    100 μl 5% saponin

    Adjust final volume to 10 ml with PBS 1×

    Store short-term at 4 °C

  5. Washing buffer

    500 μl goat serum

    500 μl 5% saponin

    Adjust final volume to 50 ml with PBS 1×

    Store short-term at 4 °C

  6. 3% PFA

    1.5 g PFA

    12.5 μl NaOH 6 M

    Adjust final volume to 50 ml with PBS 1×

    1. Combine all components in a glass bottle in a fume hood

    2. Add magnetic stirring bar

    3. Dissolve the PFA by stirring the solution on a magnetic stirrer at 56 °C in the fume hood

    4. Store aliquots at -20 °C

    5. Avoid freeze-thaw cycles

  7. 5% saponin

    2.5 g saponin

    Adjust final volume to 50 ml with distilled water

    Make aliquots and store at -20 °C

Acknowledgments

The protocol was adapted from Farrell et al. (2014). This work was supported by the Swedish Cancer Society (Cancerfonden, CAN2017/735). We acknowledge the Biochemical Imaging Center at Umeå University. We thank Wai-Lok Yau for critical reading of the manuscript.

Competing interests

The authors declare no conflicts of interest.

References

  1. Carter, L. E., Cook, D. P. and Vanderhyden, B. C. (2019). Phenotypic plasticity and the origins and progression of ovarian cancer. The Ovary(Third Edition). Leung, P. C. K. and Adashi, E. Y. (Eds.). Academic Press: 529-545.
  2. Dongre, A. and Weinberg, R. A. (2019). New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol 20(2): 69-84.
  3. Dukes, J. D., Whitley, P. and Chalmers, A. D. (2011). The MDCK variety pack: choosing the right strain. BMC Cell Biol 12(1): 43.
  4. Farrell, J., Kelly, C., Rauch, J., Kida, K., Garcia-Munoz, A., Monsefi, N., Turriziani, B., Doherty, C., Mehta, J. P. and Matallanas, D. (2014). HGF induces epithelial-to-mesenchymal transition by modulating the mammalian hippo/MST2 and ISG15 pathways. J Proteome Res 13(6): 2874-2886.
  5. Gonzalez, D. M. and Medici, D. (2014). Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal 7(344): re8-re8.
  6. Kalluri, R. and Weinberg, R. A. (2009). The basics of epithelial-mesenchymal transition. The J Clin Invest 119(6): 1420-1428.
  7. Li, C. W., Xia, W., Huo, L., Lim, S. O., Wu, Y., Hsu, J. L., Chao, C. H., Yamaguchi, H., Yang, N. K. and Ding, Q. (2012). Epithelial–mesenchymal transition induced by TNF-α requires NF-κB–mediated transcriptional upregulation of Twist. Cancer Res 72(5): 1290-1300.
  8. Song, F. N., Duan, M., Liu, L. Z., Wang, Z. C., Shi, J. Y., Yang, L. X., Zhou, J., Fan, J., Gao, Q. and Wang, X. Y. (2014). RANKL promotes migration and invasion of hepatocellular carcinoma cells via NF-κB-mediated epithelial-mesenchymal transition. PLoS One 9(9): e108507.
  9. Vidal-Quadras, M., Holst, M. R., Francis, M. K., Larsson, E., Hachimi, M., Yau, W.-L., Peränen, J., Martín-Belmonte, F. and Lundmark, R. (2017). Endocytic turnover of Rab8 controls cell polarization. J Cell Sci 130(6): 1147-1157.
  10. Yadav, A., Kumar, B., Datta, J., Teknos, T. N. and Kumar, P. (2011). IL-6 Promotes Head and Neck Tumor Metastasis by Inducing Epithelial–Mesenchymal Transition via the JAK-STAT3-SNAIL Signaling Pathway. Mol Cancer Res 9(12): 1658-1667.
  11. Yonemura, S. (2014). Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment. PLOS One 9(11): e112922.
  12. Yuan, S., Norgard, R. J. and Stanger, B. Z. (2019). Cellular plasticity in cancer. Cancer Discov. DOI: 10.1158/2159-8290.CD-19-0015.

简介

[摘要]上皮-间质转化(EMT)是上皮细胞向分化为间充质细胞的可逆过程,能够启动细胞迁移。EMT在胚胎发育,组织修复和癌症转移中起着重要作用。更好地了解EMT过程中的细胞和分子事件,不仅将为哺乳动物生物的发育以及上皮组织的再生提供新颖的见解,而且还可为癌症治疗确定新的治疗靶标。在这里,我们旨在提供有关如何在Madin-Darby犬肾脏(MDCK)II上皮细胞系中诱导EMT并在EMT诱导的细胞上进行免疫荧光染色的详细协议。


[背景]上皮细胞的特征在于细胞可塑性,即具有采用不同细胞表型的能力(Carter等,2019; Yuan等,2019)。上皮-间质转化(EMT)是上皮细胞可塑性的一种形式。在EMT期间,上皮细胞会破坏细胞间连接,使其极性失去作用,并从鳞状,长方体或柱状变为梭形并迁移,从而获得间充质细胞的特性(Kalluri和Weinberg,2009)。EMT可以通过免疫染色和测量标志物(例如E-钙粘蛋白,ZO-1,波形蛋白,纤连蛋白和N-钙粘蛋白)的表达水平进行评估(Kalluri和Weinberg,2009)。在过去十年中在EMT研究中心大多的转录因子的作用例如,SNAIL1 / 2,ZEB1 / 2,拧和微RNA(Gonzalez和奇,2014),细胞色素okines等肿瘤坏死因子-α(TNF-α)和IL-6(IL-6)(Yadav等,2011; Li等,2012),以及诸如转化生长因子β(TGF-β)信号转导和NF-κB信号转导的信号转导机制(Gonzalez和Medici ,2014; Song等,2014; Dongre和Weinberg,2019)。然而,早期EMT的调控机制以及EMT期间分子事件的精确序列仍然未知。

Madin-Darby犬肾(MDCK)上皮细胞系是研究上皮细胞极性和连接的细胞模型(Dukes等,2011; Yonemura,2014; Vidal-Quadras等,2017)。MDCK II细胞系是一株,起源于亲代MDCK细胞的更高传代(Dukes等,2011)。MDCK II细胞与亲代MDCK细胞的不同之处在于MDCK II细胞具有渗漏的细胞连接。重要的是,MDCK细胞可以在标准的2D细胞培养系统中以及在生理上更相关的3D细胞培养系统中培养(Yonemura,2014年; Vidal-Qu adras等人,2017年)。

通过用肝细胞生长因子(HGF)处理,可以很容易地在MDCK II细胞的2D细胞培养物中刺激EMT ,从而降低细胞圆度,上调波形蛋白的表达并增加与第一和第二相邻细胞的细胞距离(Farrell等人,2014)。她Ë我们提供的程序的,关于如何的详细描述中的2D细胞培养物中刺激EMT MDCK II细胞。此外,我们描述了EMT诱导的细胞对紧密连接蛋白ZO-1和经典EMT标记波形蛋白的免疫染色方法。

关键字:上皮细胞间质转型, MDCK细胞, 肝细胞生长因子, 上皮细胞可塑性, 上皮细胞间质转型, 从上皮细胞分化为间充质细胞

材料和试剂
无菌细胞培养皿,100 × 20 mm(Sarstedt,产品目录号:83.3902)
血清移液管:5毫升(Sarstedt,目录号:86.1253.001),10毫升(Sarstedt,目录号:86.1254.001),50毫升(Sarstedt,目录号:86.1256.001)
管15 ml(Sarstedt,产品目录号:62.554.502)
试管50 ml(Sarstedt,产品目录号:62.547.254)
1.5 ml微管(Sarstedt,产品目录号:72.690.301)
培养室:Lumox 8孔试样滑动可拆卸(Sarstedt的,Ç atalog号:94.6150.801)
注意:该程序也可用于8孔的Falcon TM腔室细胞培养玻片(Corning,目录号:354118)。

实验室瓶,硼硅酸盐3.3杯,50毫升(VWR,目录号:215-3261 )
实验室瓶,硼硅酸盐3.3杯,1,000毫升(VWR,目录号:215-1595 )
铝箔
ART TM Barrier专业吸管提示:10 µl(Thermo Scientific,目录号:2140),200 µl(Thermo Scientific,目录号:2770),1,000 µl(Thermo Scientific,目录号:2279)
显微镜载玻片架
盖玻片门泽尔格拉塞24 × 60毫米(赛默飞世科学,Ç atalog号:E-4137)
封口膜
MDCK II细胞系(Merck / Sigma-Aldrich,目录号:ECACC 00062107)
鹰牌最低必需培养基(EMEM),500毫升(ATCC,目录号:30-2003)
胎牛血清(FBS),500毫升(Gibco,货号:10270-106)
注意:FBS不需要热灭活。

Dulbecco的磷酸盐缓冲盐水(DPBS)1 × ,无钙,无镁,500 ml(Gibco,目录号:14190-094)
胰蛋白酶-EDTA(0.25%),酚红,100 ml(Gibco,产品目录号:25200056)
重组人HGF(衍生自HEK293),25μg(Peprotech,产品目录号:100-39H)
山羊正常血清,10 ml(Agrisera,目录号:AS10 1548)
皂苷,10克(Sigma-Aldrich,产品目录号:S4521)
1 kg多聚甲醛(PFA)(Sigma-Aldrich,产品目录号:16005)
氯化钠(NaCl),1千克(VWR C hemicals,产品目录号:27810.295 )
氯化钾(KCl),试剂级,Reag。欧元(Ph Eur),1千克(沙劳,目录号:PO02001000)
磷酸氢二钠二水合物(Na 2 HPO 4 · 2H 2 O),1千克(Scharlau,目录号:SO03391000 )
磷酸二氢钾(KH 2 PO 4 ),1千克(Merck,产品目录号:1048731000)
VECTASHIELD ®抗荧光淬灭封固介质用DAPI,将10毫升(Vector Laboratories公司,C atalog号:H-1200)
指甲油
氢氧化钠(NaOH),1千克(默克(Merck),目录号:1064691000)
一抗(表1)

表1.一抗


二抗(表2)

表2.二抗


MDCK II细胞的完整培养基(请参阅食谱)
PBS 10 × (请参阅食谱)
PBS 1 × (请参阅食谱)
阻塞缓冲区(请参见食谱)
洗涤缓冲液(请参见配方)
PFA为3%(请参阅食谱)
5%皂苷(请参阅食谱)

设备


1. Burker型计数室(Hirschmann,目录号:8100201 )     
2.通风柜     
3.的Eppendorf ®研究®加吸管,3包:0.5-10微升,10-100微升,100-1,000微升(Sigma-Aldrich公司,C atalog号:Z683884 )     
4.集成Pipetboy 2(VWR,目录号:612-0927)     
5.加热和电磁搅拌器RH basic 2(Dulova,目录号:QSA337)     
6.离心机VWR Compact Star CS4     
7. 37 °C ,5%CO 2细胞培养培养箱     
8.标准相衬显微镜     
9.共焦显微镜Zeiss 710     

软件


1. ImageJ(https://imagej.nih.gov/ij/download.html)       

程序


A.开始MDCK II细胞培养     
1.准备用于MDCK II细胞的完整培养基(请参见食谱)。     
2.用冷冻的MDCK II细胞解冻小瓶。     
3.将细胞转移到装有完整培养基的15 ml Falcon管中。     
4.离心1细胞5分钟,500转(214 ×克)。     
5.丢弃上清液。     
6.加入10 ml完全培养基。上下移液。     
7.将细胞转移到细胞培养皿中。     
8. 37℃培养细胞       °C,细胞培养箱中的5%CO 2 。


B. MDCK II细胞传代     
1.预热完全培养基和胰蛋白酶。     
2.从细胞中除去培养基。     
3.用10毫升的DPBS洗涤。     
4.加入2 ml胰蛋白酶。     
5.在37 °C下孵育细胞5分钟。     
6.在显微镜下检查细胞是否脱落。     
注意:如果细胞未分离,则继续在37 °C下孵育2-5分钟。

7.加入10 ml完全培养基。     
8.上下移液。     
9.将细胞悬浮液转移到15ml管中。     
10.将细胞以1,500 rpm(214 × g )离心5分钟。 
11.丢弃上清液。 
12.将细胞接种到新的细胞培养皿中。使用1:5的分割比例。 
13.在37 ℃下培养细胞。 

C.EMT的归纳     
第0天

在8孔室玻片中播种16,000个细胞/孔。
注意:为了在MDCK II细胞中诱导EMT,将细胞传代少于10次。

孵育细胞过夜。

第一天

1.准备HGF库存100       μg / ml并分装:

用冻干的HGF旋转小瓶。
加入250 μ的无菌BSA的1mg / ml(在PBS中)的升与HGF的小瓶中。
通过移液混合。
快下来
等分。使用无菌的1.5 ml管。
将等分试样储存在-80 °C下。
2.用完全培养基稀释HGF储备液至终浓度20 ng / ml。涡旋混合。     
3.从接种在8孔室玻片上的细胞中除去培养基。     
4.每孔加入0.5ml的DPBS。     
5.除去DPBS。     
6.加入完全含有HGF的培养基,每孔使用0.5 ml培养基。     
注意:作为对照,用不含HGF的完全培养基处理细胞。

7.将细胞在37 °C下孵育24小时。     

D.免疫荧光染色和成像     
第二天

1.解冻:     
PFA为3%(请参阅食谱)

山羊血清

5%皂苷(请参阅食谱)

2.准备:     
洗涤缓冲液(请参见配方)

阻塞缓冲区(请参见配方)

3.除去培养基。     
4.将培养物在3%PFA中固定10-15分钟。每孔使用0.5 ml PFA。     
注意:所有免疫染色步骤均在室温下进行,除非另有说明。

5.用PBS 1 ×洗涤培养物(请参阅“食谱”)。使用0.5 ml PBS 1 × /孔。     
6.每孔加入0.5 ml封闭缓冲液。在室温下孵育1小时。     
7.孵育与一抗培养物(表1) ,过夜,在4 ℃下。用封口膜覆盖文化。     
一种。以1:200的比例稀释抗ZO-1抗体。     
b。以1:500的比例稀释抗波形蛋白抗体。     
C。使用洗涤缓冲液稀释抗体。     
d。每孔最少使用200μl抗体溶液。     

第三天

8.去除抗体溶液。     
9.用PBS 1 ×洗涤。使用0.5 ml PBS 1 × /孔。     
10.用荧光二抗(表2)孵育培养物1小时。 
以1:500的比例稀释第二抗体。
使用洗涤缓冲液稀释抗体。
每孔最少使用200μl抗体溶液。
用铝箔纸覆盖培养物,以防止光照。
11.用PBS 1 ×洗涤。使用0.5 ml PBS 1x /孔。 
12.丢弃PBS 1 × 。 
13.拆下介质腔。使用拆卸装置。 
14.安装 
将几滴安装介质滴在盖玻片上。
将盖玻片幻灯片放在包含培养物的幻灯片顶部。避免气泡。
15.用指甲油密封已安装的幻灯片。 
16.将载玻片存储在4 °C的显微镜载玻片文件夹中。避光。 

第四天

用共聚焦显微镜拍摄染色细胞的图像(图1)。


数据分析


共聚焦显微镜图像ANA裂解与ImageJ软件。除了通过免疫荧光进行表型鉴定外,EMR诱导的MDCK细胞还可以通过Western印迹,细胞圆度评估以及到第一个和第二个相邻细胞的细胞距离进行分析,如Farrell等人所述。(2014)。



图1. MDCK II细胞中EMT的表型表征。A.实验方案:用肝细胞生长因子(HGF)处理MDCK II细胞24小时,固定,然后对EMT标志物染色。B.免疫染色对波形蛋白和紧密连接PROT ein- 1 (紧密连接- 1,ZO - 1 )。DAPI用于核反染。物镜20 × 。比例尺,50μm。


笔记


为了获得成功且可重复的结果,我们建议:

要严格遵守制造商关于HGF储存的准则,并避免重复进行HGF冻融循环。
应该每周在显微镜下观察MDCK II细胞,以监测表型的稳定性。
MDCK II培养物绝对不能生长过度(避免> 80%融合)。
应当定期更换细胞培养基(避免使用酸性培养基,因为酸性培养基可以诱导EMT,因此可以通过从粉红色变为黄色的颜色来识别)。

菜谱


MDCK II细胞的完全培养基
450毫升鹰牌最低必需培养基(EMEM)

50毫升胎牛血清(FBS)

PBS 10 ×
80克氯化钠           
2.2克氯化钾           
14.2克Na 2 HPO4 · 2H 2 O

2克KH 2 PO 4

用蒸馏水将最终体积调至1 L

PBS 1 ×
100毫升PBS 10 ×

900毫升蒸馏水

阻塞缓冲区
500μl山羊血清

100μl5%皂苷

用PBS将最终体积调整为10 ml 1 ×

短期存放于4 °C

洗涤缓冲液
500μl山羊血清

500μl5%皂苷

用PBS将最终体积调整为50 ml 1 ×

短期存放于4 °C

3%PFA
1.5克PFA

12.5μlNaOH 6 M

用PBS将最终体积调整为50 ml 1 ×

在通风橱中将所有组件合并到一个玻璃瓶中
加磁力搅拌棒
通过在通风橱中于56 °C的磁力搅拌器上搅拌溶液来溶解PFA
将等分试样储存在-20 °C
避免冻融循环
5%皂苷
2.5克皂苷

用蒸馏水将最终体积调至50 ml

分装并储存在-20 °C


致谢


该协议改编自Farrell等。(2014)。这项工作得到了瑞典癌症协会(Cancerfonden,CAN2017 / 735)的支持。我们感谢于默奥大学的生化成像中心。感谢丘伟乐对手稿的认真阅读。


利益争夺


作者宣称没有利益冲突。


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引用:Pastuła, A. and Lundmark, R. (2021). Induction of Epithelial-mesenchymal Transition in MDCK II Cells. Bio-protocol 11(3): e3903. DOI: 10.21769/BioProtoc.3903.
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