2.7 Cell counting
Cells were seeded onto the scaffolds at a density of
8x104 cells/well in GM. After 24 h, supernatants were
carefully collected from the scaffolds and transferred to a new tube.
The well was carefully washed with PBS, which was also transferred to
the same tube containing the supernatant. Tubes containing GM and PBS
from each individual well were centrifuged at 1000 rpm (approximately
184 g) for 5 minutes, and each pellet was resuspended in 50 µl of fresh
GM. Cells were counted using a Neubauer chamber.
Cell morphology and actin labeling
Morphology of the cells after growth in different substrates was
analyzed by scanning electron microscopy (SEM). First, the samples were
fixed in 2.5% glutaraldehyde for 6–h. The samples were rinsed with
distilled water and gradually dehydrated in two increasing series of
ethyl alcohol (35%, 50%, 70%, 85%, 95% and 100% for 15
minutes/bath). Samples were metalized with gold and visualized using a
Quanta 200 FEG SEM (FEI, Hillsboro, USA).
For actin labeling, the scaffolds or coverslips containing the cells
were washed twice with PBS and then fixed in 3.7% formaldehyde for 15
minutes at RT. The samples were then washed with PBS, permeabilized in
0.1% Triton-X100 in PBS for 10 minutes at RT, washed again with PBS,
and incubated with 0.2 µg/mL Alexa Fluor 546 phalloidin (Thermo Fisher)
in PBS for 30 minutes at RT. Next, the cells were washed with PBS and
cell nuclei were stained with DAPI (diluted to 1:1,000 in PBS) for 20
min at RT. Images were captured in a Zeiss fluorescence microscope