骨髓源内皮祖细胞胞间粘附试验

Yubo Tang; Angela Jacobi; Corina Vater; Xuenong Zou; Maik Stiehler

doi:10.21769/BioProtoc.1909

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Peer-reviewed

Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay

骨髓源内皮祖细胞胞间粘附试验

Maik Stiehler email

发布: 2016年08月20日第6卷第16期 DOI: 10.21769/BioProtoc.1909 浏览次数: 10235

评审: Xiujun FanAnonymous reviewer(s)

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Cover of Stem Cells, featuring study using the protocol.

Jun 2015

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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

Sterile serological transfer pipettes (Greiner Bio-One, catalog number: 606107 , 607107 , 760107 )
Sterile centrifuge tubes (Greiner Bio-One, catalog number: 188261 )
T75 culture flasks (Corning, catalog number: 353135 )
24-well plates (Corning, Costar^®, catalog number: 3524 )
96-well plates (Corning, catalog number: 3596 )
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)
Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190169 )
Human fibronectin (BD Biosciences, catalog number: 610077 )
Endothelial cell growth medium MV2 (ECGM2) (PromoCell GmbH, catalog number: 22121 )
Penicillin/streptomycin (5,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070-063 )
Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
Dil-ac-LDL (Thermo Fisher Scientific, catalog number: L3484 )
FITC-UEA-I (Sigma-Aldrich, catalog number: L9006 )
Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
Hoechst 33258 (Santa Cruz Biotechnology, catalog number: sc-394039 )
Calcein AM (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: L-3224 )
Ethylene diamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
Trypsin from porcine pancreas (Sigma-Aldrich, catalog number: T4799 )
Endothelial cell growth medium (see Recipes)
0.2 mg/ml Hoechst 33258 (see Recipes)
2 μM calcein-AM (see Recipes)

Equipment

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

Procedure

English

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如何引用

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.