Confirmation of differentiation parameters for SH-SY5Y cells. (a) SH-SY5Y cells were plated at concentrations of 10,000 cells or 20,000 cells per well in an xCELLigence E-plate (ROCHE) in regular DMEM media containing 10% FBS (undifferentiated cells) or in serum free DMEM containing 50 nM IGF-1 (differentiated cells). The xCELLigence system measures changes in impedance as cells attach with a readout given as cell index (CI) value. The baseline impedance is recorded using control wells containing DMEM only with no cells. Graph representative of duplicate wells and shows adhesion and proliferation over 24 hours, n = 2. A zoomed area of the first 3.5 hours shows differentiated cells attach earlier and are larger but do not proliferate. (b) SH-SY5Y cells were plated on laminin-coated glass coverslips and incubated in regular DMEM media containing 10% FBS (undifferentiated cells) or in serum free DMEM containing 50 nM IGF-1 (differentiated cells). Cells were fixed with 4% PFA, permeabilised with PHEM/0.1% Triton X, blocked with PHEM/5% goat serum. The actin cytoskeleton was stained with TRITC-phalloidin (red) and the nuclei were stained with Hoechst 33242 (blue). All images were acquired sequentially at 63× and images merged using ImageJ. Scale bar = 20 μm. Early stage differentiated cells are larger than undifferentiated cells but cell body size decreases as neurites extend in late stage differentiation. This supports the pattern seen in the xCELLigence graph.