Cell proliferation was assayed using the Vybrant MTT cell proliferation assay kit (Invitrogen) according to the protocol manual. Briefly, and equal number of NPCs (10,000 cells in triplicate) were seeded on 96 well plates coated with poly-L-ornithine hydrobromide and laminin (day 0). Cell counts were determined daily for 7 days. At the time of the assay, 100ul of medium was removed from the well and replaced with an equal volume of fresh medium without FGF2, along with 10ul of the 12 mM MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) stock solution. The cells were incubated at 37 °C for 4 h. 85 ul of medium was removed and 50 ul of DMSO was added, followed by a 10-min incubation at 37 °C. The samples were mixed well, transferred to a microplate, and the absorbance at 540 nm was determined. A total of 8 NPC lines were analyzed; 4 controls and 4 with 22q11.2 del. The fold change in cell number was compared to the day 1 pooled control samples, which was normalized to 1.0. Statistical significance of pooled controls and pooled 22q11.2 del samples was determined at each day of growth using a Student’s t-test (2-tailed).
