Biomedical Engineering Reference
In-Depth Information
carried out with a Super-dynamic Light Scattering Spectrophotometer, 'Photal' (Otsuka Elec‐
tronics) at 75 mW Ar laser.
2.9. Confocal laser scanning microscopy
pGL3 vector was labeled with PI at a PI/DNA ratio of 1:1 and particles generated with this
labeled plasmid (described in transfection protocol), were incubated with HeLa cells for 6
hours. Acidic compartments were labeled with 5μM LysoSensor, according to the instruc‐
tions provided by Molecular Probes, and membrane-bound precipitates were removed by 5
mM EDTA in PBS before observation by LEICA TCS-NT.
2.10. SDS-PAGE and Western blotting
Following generation of carbonate apatite as described above using 3 mM Ca
2+
and required
amount of fibronectin or E-cad-Fc chimera and no DNA and centrifugation at 15000 rpm for
5 min at 4
0
C, precipitated particles were washed with water with several centrifugation steps
to remove unbound proteins and dissolved with 50 mM EDTA in PBS for subsequent analy‐
sis by 7.5% SDS-PAGE in reducing condition. In order to see particle-bound fibronectin, after
SDS-PAGE, the gel was stained with Coumassie blue, washed and dried. For detection of
particle-associated E-cad-Fc, proteins after being run by SDS-PAGE were transferred to PVDF
membrane (Immobilon, Millipore) and 80 mA current was applied for 90 min to complete
transfer of the proteins. The PVDF membrane was washed with PBS (-)-containing 0.1% Tween
20 and then blocked for 1 hr at room temperature by “Blocking One” (Nacalai Tesque, Ja‐
pan). The membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-
mouse IgG for 1 hr and washed with PBS-T three to four times to completely remove non-
specific interactions. Enhanced chemiluminescence system (Amersham Bioscience) was used
for visualization.
2.11. DNA labelling, fluorescence microscopy and flow cytometry
Plasmid DNA was labelled non-covalently with propidium iodide (PI) using 1:1 weight ratio
of DNA to PI in the particle preparation medium. Labelled DNA inside the cells was ob‐
served by a fluorescence microscope (Olympus-IX71), following 4 hr incubation of differen‐
tially formulated particle suspensions with F9 cells and removing extracellularly bound particles
by 5 mM EDTA in PBS. For flow cytometric analysis using FACS Calibur (Becton, Dickinson
and Company), 1 day after transfection with pEGFP plasmid DNA, F9 cells were collected in
a sorter buffer following treatment with trypsin-EDTA and repeated centrifugation and wash‐
ing of the resulting cell pellet with PBS (-) (2 times).
3. Results and Discussion
3.1. Generation of carbonate apatite particles
Addition of only 3 mM Ca
2+
to the HCO
3
-
- buffered cell culture medium (DMEM or Wil‐
lium E, pH 7.5) and incubation at 37
0
C for 30 min, resulted in microscopically visible parti‐
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