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Amniotic

Amniotic

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Published by: Ganesh Physiovesalius on Nov 05, 2010
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11/05/2010

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ANALYTICAL SCIENCES OCTOBER 2010, VOL. 26 1
Supporting InformationAFM Studies of Cellular Mechanics during OsteogenicDifferentiation of Human Amniotic Fluid-derived Stem Cells
Qian CHEN*
 
Pan XIAO,** Jia-Nan CHEN,*
 
Ji-Ye CAI,*
Xiao-Fang CAI,*
 
HuiDING,** and Yun-Long PAN,*** Department of Chemistry, Jinan University, Guangzhou, 510632 China,**The First Affiliated Hospital of Jinan University, Guangzhou, 510632 China,
Cell culture
hAFSCs were provided by the
 
first affiliated hospital of Jinan University.
 
Cells weregrown in modified culture medium with 15% fetal bovine serum, L-glutamin, andantibiotics.
 
To differentiate hAFSCs into bone cells, we used the osteogenic inductionmedium containing 10 nM dexamethasone, 50 mM L-ascorbic acid, and 20 mM b-glycerophosphate. hAFSCs between passages 3 and 9 were used for allexperiments.
 Flow cytometry detection
Passage 3 hAFSCs were stained with various combinations of saturating amounts of monoclonal antibodies conjugated with either fluorescein isothiocyanate (FITC) or  phycoerythrin (PE) as follows: CD29—FITC, CD90—PE, CD34—PE, CD45—PE,HLA-DR—FITC, CD106-PE. Approximately 5 × 10
5
cells were analyzed by flow cytometry
.
 
 Immunostaining and confocal microscopy
To explore the cytoskeleton structure of hAFSCs after being induced for 7d, 14d, 21d,respectively, samples were fixed in 4% paraformaldehyde. Nonspecific binding sits were blocked using a 1% BSA solution for 30 min at room temperature. Intracellular actinfilaments were stained with 5 mM FITC-fhalloidin for 30 min at room temperature. Sampleswere imaged by confocal microscopy
.
 
Sample for AFM imaging 
Cells were absorbed on glass, fixed with 2.5% glutar-aldehyde for 15 min, then washed using pure water for three times. All the fixed samples were air dried. The prepared samples wereimaged at room temperature using an atomic force microscope (AFM, CP-Research) in
To whom correspondence should be addressed.E-mail: tjycai@jnu.edu.cn
2010 © The Japan Society for Analytical Chemistry

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