Trypsin edta 0.05 solution

On day 24, iPSC-differentiated cultures were dissociated and mature motor neurons were isolated using magnetic bead sorting based on L1CAM, as previously described (Saporta et al., 2015 (link)). Briefly, on day 24 of differentiation neurons were dissociated with Trypsin-EDTA 0.05% solution (Sigma) and counted. A portion of cells, called unsorted cell fraction, were separated and plated for further immunostaining characterization. The remaining neuronal culture was incubated for 1 h with anti-CD171 PE (L1CAM) antibody (Thermo Scientific) as primary antibody. Cells were centrifuged, washed once using Sorting Buffer (6% Fetal Bovine serum in 1× DPBS) and incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min. After another centrifugation and washing step, cells were ressuspended in Sorting Buffer and applied onto prepared LS Column placed into MidiMACS separator (Miltenyi Biotec), following manufacturer’s instructions. The column was washed 3 times with 3 mL of Sorting Buffer, removed from the MidiMACS separator and placed in a separated conical tube. The positive cell fraction containing the purified motor neurons was eluted with 5 mL of Sorting Buffer by firmly pushing the plunger into the column. Neuronal cultures were maintained in motor neuron maturation medium until used in end-point experiments.