For motor neuron differentiation, dissociate the stably transfected cell cultures with dissociation reagent as demonstrated to allow the cells to be collected in a 15-milliliter tube containing 5 volumes of DMEM/F12 medium. After centrifugation, resuspend the cells in human iPSC medium supplemented with micromolar ROCK inhibitor for counting.
Seed the cells on matrix-coated dishes at a 6.25 x 104 cells per centimeter squared density. For the next two days, replace the supernatants with fresh differentiation medium supplemented with doxycycline.
On day two, change the medium to Neurobasal B27 medium supplemented with gamma-secretase inhibitor, vascular endothelial growth factor receptor inhibitor, and doxycycline. On day five, dissociate the cells as demonstrated, and resuspend the detached cells in 4 milliliters of DMEM/F12 medium for counting.
Pipetting is important for a complete dissociation of the cells.
Freeze an aliquot of the motor neuron progenitors in cell freezing medium according to the manufacturer's instructions, and collect the rest of the cells by centrifugation. Resuspend the pellet in neuronal medium supplemented with 10 micromolar ROCK inhibitor.
Seed the cells on poly-ornithine laminin-coated microslide supports with polymer coverslips at a 1 x 105 cells per centimeter squared density. On day six, carefully replace the medium with fresh neuronal medium without inhibitor for culture until downstream analysis without detaching the cells from the support surface.
