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Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy
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10.1063/1.4746252
/content/aip/journal/bmf/6/3/10.1063/1.4746252
http://aip.metastore.ingenta.com/content/aip/journal/bmf/6/3/10.1063/1.4746252
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Figures

Image of FIG. 1.
FIG. 1.

Microscopic images (×20) of the C2C12 myoblasts at the first day of their seeding, and two stages (days 2 and 7) after their induction to form myotubes. The myotubes are indicated by arrows.

Image of FIG. 2.
FIG. 2.

Basic details of the DEP chamber and fluidic and electrical connections, see main text for details.

Image of FIG. 3.
FIG. 3.

Thirty electrodes (each 15 μm wide, spaced 500 μm apart, angled at 18° to the fluid flow) were located along both the top and bottom surface of the DEP chamber to form a series of fifteen 3D electrode “funnels.” The last set of five quadrupole electrodes are shown (left) with a gap of 50 μm between adjacent electrode tips. As shown (right), this facilitated the funnelling of cells deflected by negative DEP from the two outer fluid streams into the centre output fluid port.

Image of FIG. 4.
FIG. 4.

(a) Flow cytometry control experiments on cell samples (unsorted by DEP) to determine appropriate gate settings. (I) Myotubes mixed with MRC-5 GFP fibroblasts. (II) C2C12 cells co-cultured with MRC-5 GFP fibroblasts. (b) Superimposed forward scatter plots for (blue) C2C12 myoblasts (n = 59 518) and (green) MRC-5-GFP fibroblasts (n = 23,343). (c)Flow cytometry result for PI stained C2C12 myoblasts after DEP separation from myotubes, indicating 95.2% viability.

Image of FIG. 5.
FIG. 5.

(a) Flow cytometry analyses of a C2C12 sample (7 days after induction to myogenic differentiation) collected at 120 μl/h from the central exit fluid port of the DEP chamber as a function of an applied 10 V (pk-pk) voltage frequency. (b) Analyses of a sample separation at 409 kHz: (left) collection from central port (95.2% negative, 4.79% positive, for Alexa Fluor 488); (right) collection from outer flow port (1.42% negative 98.6% positive, for Alexa Fluor 488).

Image of FIG. 6.
FIG. 6.

Western blot analyses: C2C12 myoblasts and myotubes were separated by DEP at 409 kHz, and samples collected from the outer and centre fluid exit ports were blotted for embryonic myosin. The bands at ∼200 kDa (indicative of myotubes) were only present in the sample collected from the outer port, in agreement with the results shown in Figure 5. Tubulin was used to calibrate the protein loadings on the gel.

Image of FIG. 7.
FIG. 7.

(a) Flow cytometry analyses of a sample of C2C12 myoblasts co-cultured with MRC-5-GFP fibroblasts collected at 120 μl/h from the central exit fluid port of the DEP chamber as a function of an applied 10 V (pk-pk) voltage frequency. (b)Analyses of a sample separation at 500 kHz: (left) collection from central port (99.9% negative, 0.13% positive, for GFP); (right) collection from outer flow port (2.27% negative, 97.7% positive, for GFP).

Image of FIG. 8.
FIG. 8.

(a) Flow cytometry analyses of mixed sample of myotubes and MRC-5-GFP fibroblasts collected at 120 μl/h from the central exit fluid port of the DEP chamber as a function of an applied 10 V (pk-pk) voltage frequency. (b) Analyses of a sample separation at 400 kHz: (left) collection from central port (100% negative, 0.013% positive, for GFP); (right) collection from outer flow port (0.92% negative, 99.1% positive, for GFP).

Image of FIG. 9.
FIG. 9.

(a) Normalised Raman spectra of cell membranes for C2C12 myoblasts and MRC5-GFP fibroblasts. Regions 1 and 2 correspond to CH2 and CH3 bending (1440, 1460 cm−1)32,33 and CH2 stretching vibrations34,35 for saturated long CH chains (2860, 2840, and 2880 cm−1), respectively. (b) Expanded region 1. (c) Expanded region 2. These data show that the membranes of MRC5 fibroblasts have a lower proportion of saturated hydrocarbon bonds compared to C2C12 myoblasts.

Image of FIG. 10.
FIG. 10.

Scatter plot for the principal component analysis between PC3 and PC2. The PC3 loading gave the best separation of data between the three membrane types, and these are delineated by the two solid straight lines.

Image of FIG. 11.
FIG. 11.

matlab modelling of the Clausius-Mossotti polarizability factor for the three cell types, based on their estimated DEP crossover frequency values.

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/content/aip/journal/bmf/6/3/10.1063/1.4746252
2012-09-01
2014-04-20
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy
http://aip.metastore.ingenta.com/content/aip/journal/bmf/6/3/10.1063/1.4746252
10.1063/1.4746252
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