Spheroid uptake of siRNA is more efficient when complexes are formed with serum. (A) Workflow of experimental timeframe for spheroid transfection and analysis. (B) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. (C) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. White scale bar indicates 250 μM and white/black arrows indicate the location of the matrigel boundary. Summary graphs indicating the level of CTNNB1 mRNA in (D) 3D, and (E) 2D SW1463 cell cultures, at 48 hours post transfection with control or CTNNB1 siRNA complexes formed in Opti-MEM or 10% serum using RNAiMax. Levels are normalized to the housekeeping gene TATA-binding protein (TBP). Data represents mean ± 1 s.d (n = 3). Statistical significance is denoted by **P < 0.01 and ***P < 0.0001 (NS = not significant) as analyzed by a one sample t-test.