骨髓间充质干细胞粘附性测定

Zhigang Yang; Ran Xiao

doi:10.21769/BioProtoc.1895

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Peer-reviewed

Bone Marrow Mesenchymal Stem Cells Adhesion Assay

骨髓间充质干细胞粘附性测定

ZY Zhigang Yang

RX Ran Xiao email

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1895 浏览次数: 11047

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Sep 2015

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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, Invitrogen^TM, catalog number: 11965092 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10099141 )
Trypsin (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 25200-056 )
Soybean trypsin inhibitor (Thermo Fisher Scientific, Invitrogen^TM, 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, Forma^TM, catalog number: 3111 )
Microscope (Nikon, model: ECLIPSE TE2000-S )
Microplate reader (ThermoFisher Scientific, model: Multiscan MK3 ) or spectrometer with 550 nm wavelength available.