Accumax

Cells were sorted using a BD FACSARIA III (BD Biosciences, San Jose, CA). Single-cell suspensions of EBs in 96-well plates were obtained by collecting all EBs of one 96-well plate in one 15 ml tube, centrifugation at 1500 RPM for 2 min, followed by removal of 14 ml of supernatant. Then, 0.5 ml of Accumax (Chemicon, Germany) was added to the EBs in 0.5 ml of medium and the contents were mixed with a 1 ml pipette and incubated for 15 min at room temperature. For Neph3-positive cells isolation and measurement, EBs were dissociated with Accumax as described above on 11, 13, 15 and 17 DIV and filtered through a cell strainer (BD Biosciences). Isolated cells were incubated in Neph3 monoclonal antibody (30 min, Sheep 1:200, R&D Systems) and labeled with a PE-conjugated secondary antibody (30 min, BD Biosciences). Doublets were excluded from the analysis. Sorted cells were gathered in ice-cold DMEM/F12/N2/10%FCS or NPCs medium and aggregated again in low-cell adhesion 96-well culture plates or 24-well plates.