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Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay
骨髓源内皮祖细胞胞间粘附试验   

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Abstract

Cell-cell adhesion ensures tight contacts between neighbouring cells, which is necessary for cell segregation, as well as for the morphological and functional differentiation of different tissues. Evidently there are cell-cell recognition systems that make cells of the same type preferentially adherent to one another. Homotypic cell adhesion is particularly important in mediating a range of physiological processes such as cell survival, migration and remodeling of vessels. Thus in the present study we selected two populations of endothelial progenitor cells which are from the same donor to investigate the possible effect of a small molecule compound Icariin on homotypic cell adhesion. Many angiogenic factors can destabilize the organization of intercellular junctions, causing endothelial barrier opening. In the present study, we observed that Icariin treatment reduced the level of VE-cadherin expression in EPCs indicating a decrease in cell-cell adhesion-a proof of the pro-angiogenic effect of Icariin. In summary, the observed loss of homotypic adhesion of EPCs may contribute to the enhanced angiogenic effect exerted by Icariin.

Keywords: Endothelial progenitor cell(内皮祖细胞), Intercellular adhesion(细胞间黏附), Cell differentiation(细胞分化)

Materials and Reagents

  1. Sterile serological transfer pipettes (Greiner Bio-One, catalog number: 606107 , 607107 , 760107 )
  2. Sterile centrifuge tubes (Greiner Bio-One, catalog number: 188261 )
  3. T75 culture flasks (Corning, catalog number: 353135 )
  4. 24-well plates (Corning, Costar®, catalog number: 3524 )
  5. 96-well plates (Corning, catalog number: 3596 )
  6. Human bone marrow-derived endothelial progenitor cells (BM-EPCs) [isolated from bone marrow of healthy donors using Ficoll-Paque density gradient (1.077 g/cm3)] (Tang et al., 2015)
  7. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190169 )
  8. Human fibronectin (BD Biosciences, catalog number: 610077 )
  9. Endothelial cell growth medium MV2 (ECGM2) (PromoCell GmbH, catalog number: 22121 )
  10. Penicillin/streptomycin (5,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
  11. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  12. Dil-ac-LDL (Thermo Fisher Scientific, catalog number: L3484 )
  13. FITC-UEA-I (Sigma-Aldrich, catalog number: L9006 )
  14. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  15. Hoechst 33258 (Santa Cruz Biotechnology, catalog number: sc-394039 )
  16. Calcein AM (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L-3224 )
  17. Ethylene diamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  18. Trypsin from porcine pancreas (Sigma-Aldrich, catalog number: T4799 )
  19. Endothelial cell growth medium (see Recipes)
  20. 0.2 mg/ml Hoechst 33258 (see Recipes)
  21. 2 μM calcein-AM (see Recipes)

Equipment

  1. Horizontal centrifuge (Eppendorf, catalog number: 5702RH )
  2. CO2 cell culture incubator (Thermo Fisher Scientific, catalog number: 51026280 )
  3. Flow cytometer (LSR II) (BD Biosciences, catalog number: 342975 )
  4. Scepter 2.0H handheld automated cell counter, w/50 pk of 60 µM Sensors (Merck, Merck Millipore, catalog number: PHCC20060 )
  5. Biological safety cabinets (Thermo Fisher Scientific, catalog number: 51026639 )
  6. Inverted fluorescence microscope (Zeiss, catalog number: Apotome.2 )

Procedure

  1. Bone marrow sample was diluted with equal volume of DPBS, then the diluted sample was carefully layered onto the top of Ficoll-Paque PLUS. Centrifuge at 400 x g for 30 min with the brake off (Figure 1).
  2. The mononuclear cells were carefully harvested from the interface between the Ficoll-Paque PLUS and plasma using a sterile Pasteur pipette, and transfered to sterile centrifuge tubes.
  3. Cells were washed with an equal volume of DPBS twice to remove the Ficoll-Paque PLUS residue. After washing, the isolated mononuclear cells were cultivated in flasks coated with human fibronectin (25 μg/ml) and induced by ECGM2 medium at 37 °C with 5% CO2 in humidified air at a density of 3-5 x 106/cm2 (Figure 2).
  4. After 72 h in culture, non-adherent cells were removed by washing with phosphate-buffered saline (PBS), preheated fresh ECGM2 medium was applied and the cultivation was maintained through 7 d. All cells used in this experiment are cultivated with ECGM2 at passages 3 to 5.
  5. Quantitative fluorescence-activated cell sorting (FACS) was performed on a vantage SE flow cytometer to detect the surface marker of CD34, CD133, KDR, VE-cadherin, E-selectin, vWF on the cells at 7 and 14 d.
  6. Immunofluorescence staining was performed using Dil-ac-LDL and FITC-UEA-I. Brifely, cells were first incubated with Dil-ac-LDL (5 μg/ml) at 37 °C for 2 h and then fixed through incubation with 4% paraformaldehyde for 20 min. They were subsequently incubated with UEA (10 μg/ml) at 37 °C for 1 h. After washing with PBS, the cells were visualized and photographed using a fluorescence microscope and counted at 200x magnification. Double-positive cells were considered to be differentiating EPCs.
  7. Plate 5 x 103 BM-EPCs in a 96-wells plate overnight to a confluent monolayer in ECGM2.
  8. Label the cells with 10 μg/ml Hoechst 33258 dye in PBS for 30 min at 37 °C, followed by washing with PBS three times. After washing, the cells are kept in the PBS in the cell incubator until the inoculating of another set of cells.
  9. Another set of BM-EPCs from the same donor are starved in a 96-wells plate (5 x 103/well) with basic medium (without any supplemented growth factors or fetal bovine serum) for 24 h, and treated with small molecule compounds.
  10. After 48 h incubation with the compounds to be investigated, the cells are washed twice with PBS, incubated with 2 μM calcein AM (100 μl/well) for 1 h in the cell incubator at 37 °C, followed by washing with PBS three times.
  11. The cells are detached using 0.25% trypsin-0.02% EDTA (50 μl/well) for 3 min, then resuspended with 100 μl ECGM2 and plated onto the established cell monolayer (acceptor cells) with the density of 3 x 103/well.
  12. The cultures were incubated in the PBS for 20 min at 37 °C in a 5% CO2 cell culture incubator.
  13. After that the cultures are washed gently using PBS (100 μl/well) three times.
  14. Attachment and spreading of the plated cells are monitored and recorded with a fluorescence microscope every 5 min (Figure 3).
  15. Quantification of intercellular adhesion is performed by counting the number of cells that are calcein-AM positive per microscopic field of view.   

Representative data



Figure 1. Images of cells collected from the interface between the Ficoll-Paque PLUS and plasma. A. Bone marrow sample was diluted with equal volume of DPBS, then the diluted sample was carefully layered onto the top of Ficoll-Paque PLUS. B. After centrifuging at 400 x g for 30 min, the mononuclear cells were harvested from the interface between the Ficoll-Paque PLUS and plasma.


Figure 2. Light microscope image of mononuclear cells. The mononuclear cells were carefully harvested from the interface between the Ficoll-Paque PLUS and plasma using a sterile Pasteur pipette, and transfered to sterile centrifuge tubes. After washing, the isolated mononuclear cells were cultivated in flasks coated with human fibronectin (25 μg/ml) and induced by ECGM2 medium at 37 °C with 5% CO2 in humidified air at a density of 3-5 x 106/cm2.


Figure 3. Fluorescence microscope images of cells after intercellular adhesion assay. A. Cells were plated overnight to a confluent monolayer in ECGM2, and labeled the cells with Hoechst 33258. B. Representative fluorescence micrograph demonstrating cells after intercellular adhesion assay.

Notes

  1. This protocol can be used for other cell lines, however we recommend a pilot experiment to find optimal conditions for any cell line used. These include:
    1. Optimal cell density, which is dictated by several parameters such as cell size (the larger the cells, the lower the density) and cell adhesive ability.
    2. Time of attachment. Weaker adherent cell lines may require longer incubation time to track adhesive formation.

Recipes

  1. Endothelial cell growth medium
    0.02 ml/ml fetal calf serum
    5 ng/ml epidermal growth factor (recombinant human)
    10 ng/ml basic fibroblast growth factor (recombinant human)
    20 ng/ml insulin-like growth factor (Long R3 IGF)
    0.5 ng/ml vascular endothelial growth factor 165 (recombinant human)
    1 μg/ml ascorbic acid
    22.5 μg/ml heparin
    0.2 μg/ml hydrocortisone
    100 U/ml of penicillin/streptomycin
  2. 0.2 mg/ml Hoechst 33258
    10 mg Hoechst 33258
    50 ml of sterile PBS
    Keep this stock solution at 4 °C in shelters from light.
    Note: The stock solution can only be kept for 30 d.
  3. 2 μM calcein-AM
    Add 5 µl of the supplied 4 mM calcein-AM stock solution to 10 ml of sterile, tissue culture-grade D-PBS, vortexing to ensure thorough mixing. This gives an approximately 2 µM calcein-AM solution.
    Note: This staining solution should be freshly prepared each time. The stock solution must be kept at -20 °C and protected from light.

Acknowledgments

The concept of this protocol was adapted from our previous study, in which the intercellular adhesion assay was used to measure cell-cell adhesion ability of endothelial progenitor cells (Tang et al., 2015).This study was supported by the German Academic Exchange Service and Federal Ministry of Education and Research (D/09/04774).

References

  1. Tang, Y., Jacobi, A., Vater, C., Zou, L., Zou, X. and Stiehler, M. (2015). Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells. Stem Cells 33(6): 1863-1877.

简介

细胞粘附保证了相邻细胞之间的紧密接触,这对于细胞分离以及不同组织的形态和功能分化是必要的。显然,存在使相同类型的细胞优先彼此粘附的细胞 - 细胞识别系统。同型细胞粘附在介导一系列生理过程例如细胞存活,迁移和血管重塑中特别重要。因此,在本研究中,我们选择两个来自相同供体的内皮祖细胞群体,以研究小分子化合物Icariin对同型细胞粘附的可能影响。许多血管生成因子可以破坏细胞间连接的组织,导致内皮屏障开放。在本研究中,我们观察到Icariin治疗减少EPC中VE-钙粘蛋白表达的水平,表明细胞 - 细胞粘附减少 - 证明Icariin的促血管生成作用。总之,所观察到的EPCs的同型粘附的丧失可能有助于由Icariin施加的增强的血管生成作用。

关键字:内皮祖细胞, 细胞间黏附, 细胞分化

材料和试剂

  1. 无菌血清学转移移液管(Greiner Bio-One,目录号:606107,607107,760107)
  2. 无菌离心管(Greiner Bio-One,目录号:188261)
  3. T75培养瓶(Corning,目录号:353135)
  4. 24孔板(Corning,Costar ,目录号:3524)
  5. 96孔板(Corning,目录号:3596)
  6. 人骨髓来源的内皮祖细胞(BM-EPC)[使用Ficoll-Paque密度梯度(1.077g/cm 3)从健康供体的骨髓分离](Tang等,/em>。,2015)
  7. Dulbecco's磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM ,目录号:14190169)
  8. 人纤连蛋白(BD Biosciences,目录号:610077)
  9. 内皮细胞生长培养基MV2(ECGM2)(PromoCell GmbH,目录号:22121)
  10. 青霉素/链霉素(5,000U/ml)(Thermo Fisher Scientific,Gibco< sup>,目录号:15070-063)
  11. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  12. Dil-ac-LDL(Thermo Fisher Scientific,目录号:L3484)
  13. FITC-UEA-I(Sigma-Aldrich,目录号:L9006)
  14. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  15. Hoechst 33258(Santa Cruz Biotechnology,目录号:sc-394039)
  16. Calcein AM(Thermo Fisher Scientific,Molecular Probes TM ,目录号:L-3224)
  17. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  18. 来自猪胰腺的胰蛋白酶(Sigma-Aldrich,目录号:T4799)
  19. 内皮细胞生长培养基(参见配方)
  20. 0.2mg/ml Hoechst 33258(参见配方)
  21. 2μM钙黄绿素-AM(参见配方)

设备

  1. 水平离心机(Eppendorf,目录号:5702RH)
  2. CO 2细胞培养箱(Thermo Fisher Scientific,目录号:51026280)
  3. 流式细胞仪(LSR II)(BD Biosciences,目录号:342975)
  4. Scepter 2.0H手持式自动细胞计数器,w/50 pk 60μM传感器(Merck,Merck Millipore,目录号:PHCC20060)
  5. 生物安全柜(Thermo Fisher Scientific,目录号:51026639)
  6. 倒置荧光显微镜(Zeiss,目录号:Apotome.2)

程序

  1. 用等体积的DPBS稀释骨髓样品,然后将稀释的样品小心地铺在Ficoll-Paque PLUS的顶部。在400离心机离心30分钟,制动器关闭(图1)。
  2. 使用无菌巴斯德移液管从Ficoll-Paque PLUS和血浆之间的界面小心收获单核细胞,并转移到无菌离心管中。
  3. 用等体积的DPBS洗涤细胞两次以除去Ficoll-Paque PLUS残余物。洗涤后,将分离的单核细胞培养在用人纤连蛋白(25μg/ml)包被的烧瓶中,并在37℃下用5%CO 2在潮湿空气中的密度为3的ECGM2培养基-5×10 6 /cm 2 (图2)
  4. 培养72小时后,通过用磷酸盐缓冲盐水(PBS)洗涤除去非贴壁细胞,应用预热的新鲜ECGM2培养基,并维持培养7天。在本实验中使用的所有细胞在第3至5代用ECGM2培养
  5. 在Vantage SE流式细胞仪上进行定量荧光激活细胞分选(FACS),以在7天和14天检测细胞上CD34,CD133,KDR,VE-钙粘蛋白,E-选择素,vWF的表面标志物。
  6. 使用Dil-ac-LDL和FITC-UEA-I进行免疫荧光染色。简言之,细胞首先与Dil-ac-LDL(5μg/ml)在37℃孵育2小时,然后通过与4%多聚甲醛孵育20 min。随后将它们与UEA(10μg/ml)在37℃下温育1小时。用PBS洗涤后,使用荧光显微镜观察细胞并拍照,并以200x放大倍数计数。双阳性细胞被认为是区分EPC。
  7. 将5×10 3个BM-EPC在96孔板中过夜,以在ECGM2中汇合为单层。
  8. 在37℃下用10μg/ml Hoechst 33258染料在PBS中标记细胞30分钟,然后用PBS洗涤三次。洗涤后,将细胞保存在细胞培养箱中的PBS中,直到接种另一组细胞
  9. 来自相同供体的另一组BM-EPC在具有碱性介质(不含任何补充的生长因子或胎牛血清)的96孔板(5×10 3个/孔)中饥饿24小时,并用小分子化合物处理
  10. 与待研究的化合物孵育48小时后,用PBS洗涤细胞两次,在37℃下在细胞培养箱中用2μM钙荧光素AM(100μl/孔)温育1小时,随后用PBS洗涤三次。
  11. 使用0.25%胰蛋白酶-0.02%EDTA(50μl/孔)将细胞分离3分钟,然后用100μlECGM2重悬,并以3×10 3个细胞的密度接种到建立的细胞单层(受体细胞)上 /well。
  12. 将培养物在PBS中在37℃下在5%CO 2细胞培养箱中温育20分钟。
  13. 之后,使用PBS(100μl/孔)轻轻洗涤培养物三次
  14. 用荧光显微镜每隔5分钟监测和记录电镀细胞的附着和铺展(图3)
  15. 通过计数每个微观视野中钙黄绿素-AM阳性的细胞数量来进行细胞间粘附的定量。

代表数据



图1.从Ficoll-Paque PLUS和血浆之间的界面收集的细胞的图像。 A.用等体积的DPBS稀释骨髓样品,然后将稀释的样品小心地分层到Ficoll-Paque PLUS。 B.在400×g离心30分钟后,从Ficoll-Paque PLUS和血浆之间的界面收获单核细胞。


图2.单核细胞的光学显微镜图像。使用无菌巴斯德移液管从Ficoll-Paque PLUS和血浆之间的界面小心地收获单核细胞,并转移到无菌离心管中。洗涤后,将分离的单核细胞培养在用人纤连蛋白(25μg/ml)包被的烧瓶中,并在37℃下用5%CO 2在潮湿空气中的密度为3的ECGM2培养基-5×10 6 /cm 2

图3.细胞间粘附测定后细胞的荧光显微镜图像。A.将细胞过夜铺板到ECGM2中的汇合单层,并用Hoechst 33258标记细胞。B.代表性荧光显微照片,显示细胞间的细胞粘附测定。

笔记

  1. 这个协议可以用于其他细胞系,但我们建议一个试点实验找到使用任何细胞系的最佳条件。 这些包括:
    1. 最佳细胞密度,由细胞大小(细胞越大,密度越低)和细胞粘附能力等几个参数决定。
    2. 附着时间。 较弱的贴壁细胞系可能需要较长的温育时间以跟踪粘合剂形成。

食谱

  1. 内皮细胞生长培养基
    0.02ml/ml胎牛血清 5ng/ml表皮生长因子(重组人) 10ng/ml碱性成纤维细胞生长因子(重组人) 20ng/ml胰岛素样生长因子(Long R3 IGF) 0.5ng/ml血管内皮生长因子165(重组人) 1μg/ml抗坏血酸
    22.5μg/ml肝素
    0.2μg/ml氢化可的松
    100U/ml青霉素/链霉素
  2. 0.2mg/ml Hoechst 33258
    10mg Hoechst 33258
    50ml无菌PBS
    将此储备液保存在4°C避光避光。
    注意:储备溶液只能保存30天。
  3. 2μM钙黄绿素-AM 加入5微升提供的4毫米钙黄绿素AM储备液到10毫升无菌,组织培养级D-PBS,涡旋,以确保充分混合。 这产生约2μM钙黄绿素-AM溶液 注意:该染色溶液应每次新鲜制备。 储备溶液必须保存在-20°C,避光。

致谢

该方案的概念改编自我们以前的研究,其中细胞间粘附测定用于测量内皮祖细胞的细胞 - 细胞粘附能力(Tang,等人,2015)。本研究 得到了德国学术交流服务和联邦教育和研究部(D/09/04774)的支持。

参考文献

  1. (a),(b),(c),(c),(c),(c),(d),(d),(d) Icariin促进血管生成分化并防止内皮祖细胞中的氧化应激诱导的自噬。 33(6):1863-1877。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tang, Y., Jacobi, A., Vater, C., Zou, X. and Stiehler, M. (2016). Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay. Bio-protocol 6(16): e1909. DOI: 10.21769/BioProtoc.1909.
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