26 2010 © The Japan Society for Analytical Chemistry


Supporting AFM Studies of

Information Mechanics during Osteogenic


Differentiation of Human Amniotic Fluid-derived Stem Cells
Qian CHEN* Pan XIAO,** Jia-Nan CHEN,* DING,** and Yun-Long PAN,** Ji-Ye CAI,*† Xiao-Fang CAI,* Hui

* 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 were grown in modified culture medium with 15% fetal bovine serum, L-glutamin, and antibiotics. To differentiate hAFSCs into bone cells, we used the osteogenic induction medium containing 10 nM dexamethasone, 50 mM L-ascorbic acid, and 20 mM b-glycerophosphate. hAFSCs between passages 3 and 9 were used for all experiments.
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 × 105 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 actin filaments were stained with 5 mM FITC-fhalloidin for 30 min at room temperature. Samples were 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 were imaged at room temperature using an atomic force microscope (AFM, CP-Research) in

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contact mode in air. Measurement of cell elasticity using the AFM The prepared samples were observed using AFM (CP-Research, Veeco, USA). Images were acquired at room temperature in the contact mode in air and the AFM-based force spectroscopy was used to perform the force detection. All force–distance curve experiments were performed at the same loading rate. The curvature radius of the silicon tip is about 10 nm, as for the tips used in the contact mode, the length, width and thickness of cantilever are115, 30, 3.5 mm, respectively, the oscillation frequency is 255 kHz, and the force constant is 0.01N/m (manufacturer offered). A piezoelectric scanner with a maximum XY range of 100 ×100 μm, and all of the images were flattened with the provided software to eliminate low-frequency background noise in scanning direction. An isolated cell with normal morphology was identified using the optical microscope and the AFM cantilever probe was positioned over the cytoplasmic region of the cell between the nucleus and the cell peripheral margin. Each cell was mechanically probed with AFM at several locations over a 5 × 5μm area, avoiding the cell’s perinuclear region. The force curve was obtained by measuring the cantilever deflection at every vertical z-position of the cantilever as it approached and indented the cell. The indentation depth is 10% of the total cell height. A total of ~20 cells of each type and experimental condition were used, with ~50 force-distance curves acquired from each cell. The force-distance curves were collected and analyzed according to the Hertz model, as shown in the following equation:
F= 4 E 3 1-ν 2 Rδ3

Where ν is the Poisson ratio, F is the loading force which can be obtained from the force-distance curves, δ is the indentation, which is 10% of the total cell height, was considered for the calculation to limit the influence of the underlying substrate. E is the Young’s modulus, and R is the radius of the curvature of the AFM tip, respectively. The cellular Poisson’s ratio was assumed to be 0.5, which treats the cell as an incompressible material.

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