Problem	Possible Cause	Solution
EB survival is low after passaging	Pipetting too vigorously to remove cells from plates Cells are being aspirated with the supernatant	Leave dispase on hPSCs longer to ensure easy detachment of cells Allow cells to settle for at least 5 minutes by gravity or centrifuge for 1 minute at 800 RPM
Neurosphere yield is low	Cells were not plated densely enough with FBS	Increase density upon next plating
Cells are not adhering to plates with 10% FBS	Too much NIM was added to the well resulting in less than 10% FBS in the final volume	Instead of changing the medium, add 5% more FBS to each well and return to incubator overnight. Change medium the following day and add fresh NIM to each well
Undifferentiated cells growing too slowly	Cells were broken up too much during passaging	At next passage, pipet cells fewer times to break up cell aggregates and increase this number with each subsequent passage
Colonies are taking 15–20 minutes to detach from plate	Dispase is not warm Dispase powder did not completely dissolve when solution was made	Allow dispase to warm in 37°C water bath for 15 min When making dispase, add powder to warm DMEMF/12 and allow enzyme to dissolve at 37° for at least 20 minutes
Recently plated undifferentiated hPSCs or EBs are not evenly distributed across the well	Plates were not agitated when placed in the incubator or were disturbed by opening/closing of incubator door	Agitate plates front/back and side/side in short, quick movements to ensure an even distribution of cells on the well surface. Place plates near the back of the incubator to minimize disturbance by opening/closing of incubator door