Bone Marrow Mesenchymal Stem Cells Adhesion Assay
骨髓间充质干细胞粘附性测定
发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1895 浏览次数: 11738
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
Abstract
Mesenchymal stem cells (MSCs) are widespread in adult organisms and involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. As cell adhesion play important roles in cell interactions and signaling, to thoroughly evaluate the adhesion ability of MSCs is of vital importance to clarify the mechanism of self-renewal, proliferation, activation and migration of MSCs in different microenvironments. Based on the method by Siler et al., 2000, we revised the protocol in order to provide details on how to evaluate the adhesion ability of MSCs from bone marrow (BMSCs) on extracellular matrix (ECM) protein laminins. The current protocol can also be easily translated to MSCs with other treatments or ECMs such as collagens, fibronectin, etc.
Keywords: Mesenchymal stem cells (间充质干细胞)
Extracellular matrix (细胞外基质)
Adhesion (粘附)
Stem cells (干细胞)
Laminin (层粘连蛋白)
Materials and Reagents
- 96 well culture plate (Sigma-Aldrich, Corning® Costar®, catalog number: CLS3595 )
- Human bone marrow derived MSCs, isolated and cultured as previously described (Kern et al., 2006)
Note: Cells in the third cell passage were used. - Laminin 511&521 (Biolamina, catalog number: LN511 ; LN521 )
- DMEM (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11965092 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10099141 )
- Trypsin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25200-056 )
- Soybean trypsin inhibitor (Thermo Fisher Scientific, InvitrogenTM, catalog number: 17075-029 )
- BSA (Sigma-Aldrich, catalog number: V900933 )
- Blocking buffer (0.5% BSA in PBS without Ca2+ and Mg2+)
- Wash buffer (0.1% BSA in PBS without Ca2+ and Mg2+)
- 2% SDS (Sigma-Aldrich, catalog number: 71729 )
- 4% paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
- NaCl (Sigma-Aldrich, catalog number: S7653 )
- KCl (Sigma-Aldrich, catalog number: 746436 )
- Na2HPO4 (Sigma-Aldrich, catalog number: 795410 )
- KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
- CaCl2·2H2O (Sigma-Aldrich, catalog number: 223506 )
- MgCl2·6H2O (Sigma-Aldrich, catalog number: M9272 )
- HCl (Sinopharm chemical reagent Being Co.ltd, catalog number: 10011008 )
- Crystal violet powder (Sigma-Aldrich, catalog number: C6158 )
- Ethanol (Sinopharm chemical reagent Being Co.ltd, catalog number: 10009159 )
- PBS with/without Ca2+ and Mg2+ (see Recipes)
Note: Commercial DPBS with/without calcium and magnesium can also be used. - 0.1% crystal violet staining solution (see Recipes)
Equipment
- Small shaker for microtiter plate (IKA, model: MS3 Digital )
- Scanner (UMAX, model: POWERLOOK 2100XL )
- Series II 3110 Water-Jacketed CO2 chamber (Thermo Fisher Scientific, FormaTM, catalog number: 3111 )
- Microscope (Nikon, model: ECLIPSE TE2000-S )
- Microplate reader (ThermoFisher Scientific, model: Multiscan MK3 ) or spectrometer with 550 nm wavelength available.
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yang, Z. and Xiao, R. (2016). Bone Marrow Mesenchymal Stem Cells Adhesion Assay. Bio-protocol 6(15): e1895. DOI: 10.21769/BioProtoc.1895.
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分类
干细胞 > 成体干细胞 > 间充质干细胞
细胞生物学 > 细胞运动 > 细胞运动性
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