Figure 5. Schematic diagram demonstrating strategy of drug treatments in sphere-formation assay.

2 × 10³ of U251 or SH-SY5Y cell suspensions are suspended in Matrigel™/serum free DMEM (1:1) and plated gently around the rim of individual wells of a 24-well plate. Media (alone or with drug) is added gently to the center of each well and changed every 2–3 days. Spheres are formed and serially passaged without treatment for 5 generations (G) to enrich for cancer stem cells, and then drugs are added at G5 to potentially target those cells; or treatment is added to every generation and then spheres are serially propagated to investigate whether the effect is permanent or reversal. The SFU, expressed as %, is calculated by dividing the number of spheres counted by the number of input cells (2 × 10³ cells) and then multiplied by 100.