Secretion and orientation of matrix proteins by C2C12 cells under serum free conditions on etched glass.
(a) C2C12 cells (3500/cm2) were seeded on un-etched and etched glass coverslips in the wells of a 12-well plate and cultured for 3 days in serum free proliferation medium. After 3 days, the media was changed to a serum free differentiation medium and cells were cultured for another 6 days. Post differentiation, cells were fixed and stained with anti-myosin mouse monoclonal (NOQ7.5.4D) antibody. The secondary antibody used was a goat anti-mouse alexafluor 488 antibody. Scale bar—100 μm. (b) The expression of matrix proteins by C2C12 myoblasts was examined on etched coverslips 3.5 hours after plating and in (c) undifferentiated (day 3 proliferation) and differentiated (day 6 differentiation) cell cultures. At these time-points cells were fixed and the coverslips immunostained using antibodies recognising perlecan, collagen I, collagen IV and fibronectin (all rabbit polyclonal antibodies.) The secondary antibody was goat anti-rabbit alexafluor 488 antibody. Nuclei are stained with DAPI (blue). Scale bar—50 μm.
