Analyzing C2C12 Cell Morphology on Nanofibers

The morphology of the C2C12 cells cultivated onto CA and CA@A nanofibers was determined by i) SEM and ii) F-actin staining.
For SEM analysis, C2C12 cells were seeded onto CA and CA@A nanofibers and cultivated at 37°C and 5% CO₂ in GM. After 2 and 7 days of culturing, samples were fixed in 2.5% glutaraldehyde for 6 h at RT. Samples were then rinsed with distilled water and gradually dehydrated in two increasing series of ethyl alcohol (35%, 50%, 70%, 85%, 95% and 100% for 15 min/bath). Samples were metalized with gold and visualized using a Quanta 200 FEG SEM (FEI, Hillsboro, USA).
For F-actin staining, C2C12 cells were seeded onto CA and CA@A nanofibers and cultivated at 37°C and 5% CO₂ for 7 days in GM. After washing in PBS, cells were fixed in 3.7% formaldehyde for 15 min at RT. Samples were permeabilized in 0.1% Triton-X100 in PBS for 10 min at RT, washed with PBS, and incubated with 0.2 μg/mL Alexa Fluor 546 Phalloidin (Thermo Fisher) in PBS, for 30 min at RT. Next, cell nuclei were counterstained with DAPI (diluted to 1:1,000 in PBS) for 20 min at RT. Images were obtained in a Zeiss fluorescence microscope.