A predefined number of cells was seeded in suspension culture plates (Greiner Bio-One, Kremsmünster, Austria) 24 h after transfection. Cells were then cultivated with Spheromax CSC Medium (Promocell, Heidelberg, Germany). After 7 days, spheres were counted (number per microscopic field of view, 50× magnification) and the area was measured using an open source ImageJ macro, manually correcting visually incorrect counts [65 (link)].
Free full text: Click here