Problem	Possible reason	Solution
Recovery of too many dead cells	Time delay in preparing the brain before enzymatic treatment	Make sure the dissection set up and media are ready before sacrificing the fish (also check sterility, temperature)
Stringent papain treatment	Mechanical dissociation needs to be delicate enough to generate a live single cell suspension, but strong enough to avoid leaving behind too many clumps of tissue.  Respect the recommended times and temperatures for incubation/centrifugation periods
Neurospheres do not appear or grow poorly	Incorrect temperature	Check that incubator set at 30 °C is providing the correct temperature.
Growing spheres removed with debris	For each well, the aspiration of debris has to be performed well on top of the medium. Avoid entering into the medium with your pipet
The integrity of some reagents (B27 supplement, EGF, FGF) is weakened	This integrity constitutes limiting factors in cell growth. Check batch number, and the way samples were preserved. Avoid thaw/refreeze cycles of any sampled reagent used in culture
Presence of single cells	Mode of transfer/expansion of neurospheres is too harsh	This step is an expansion/dilution step because too many spheres are formed in the well. Avoid harsh sphere collection during transfer to prevent neurosphere dissociation into single cells
Absence of cells on matrigel	Matrigel was not properly thawed/diluted	Matrigel from -20 °C needs at least 30 min at 4 °C to thaw and remains viscous Pipet carefully
No adhesion on matrigel	The volume of cell suspension has to be small enough to allow cell deposition on matrigel coat
Allow more time for cell deposition (up to 2 hr if larger volumes are used)
Fresh differentiation medium too cold	When replacing the cell-attachment medium, the differentiation medium needs to be warmed up at 30 °C to prevent thermal shock on the deposited cells
Poor nucleofection	Dead cells	Make sure that you do not generate air bubbles while performing nucleofection. Use high quality DNA vectors by using Maxi-preparations. At least 1 - 2 hr after nucleofection, replace culture medium using either fresh Z-differentiation condition medium or Z-condition medium.
Few positive neurospheres	Neurospheres of varying sizes can lead to poor nucleofection. Using neurospheres at DiV2 and DiV3 are ideal for nucleofection. Density of cells should not exceed 4 x 103 neurospheres ml-1 in a 100 ml reaction.