Cells were resuspended in HEPES buffer containing the following (in mm): 136 NaCl, 5.6 KCl, 1 MgCl2, 2.2 CaCl2, 11 glucose, 10 HEPES; pH adjusted to 7.4 with NaOH before plating on poly - d - lysine (0.02 mg /
ml; BD Bioscience) and
laminin (0.02 mg /
ml; Sigma - Aldrich)- coated coverslips.
and cultured in 5 % O2 / 7 % CO2 in Bottenstein - Sato F12 medium with 10 ng /
ml human recombinant basic fibroblast growth factor (Peprotech) on a substrate of 1 µg / cm2 fibronectin (Chemicon) and 0.5 µg / cm2
laminin (Invitrogen).