Table 3:

Troubleshooting

Step	Problem	Possible Reason	Solution
1A(ii)	mESCs are not reaching confluency at the predicted time	- Need to seed a different ratio at passage	- The passage ratio can be changed in order to increase cell density or shift when passaging needs to be conducted. Passage at a 1:3 ratio to achieve confluence after 1 day. Passage at a 1:8 ratio to achieve confluence after 3 days.
1B(ii)	hPSCs not reaching confluency	- Initial seeding density is too low	- Try a few different seeding densities when working with a new cell line.
2B(vii)	Cell layers are detaching from plate during human differentiation	- Media is too cold - Re-feeding is dislodging the layers - Incompatible Matrigel lot - Inefficient Matrigel coating	- Make sure media is at least at room temperature before feeding. - Make sure the pipette dispenser is set to the slowest setting before feeding. - Test the differentiation with different Matrigel lots. - Use fresh Matrigel and be sure to coat for 24 hours.
3A(i)	Not enough V2a interneurons after selection for subsequent experiments	- Need to induce more V2a interneurons	- To obtain the necessary high seeding density, multiple differentiation plates may need to be pooled together depending on the surface for replating or aggregation.
4B(iii)	Cell layers ball up during maturation	The cells have been on the same plate for too long and may be losing the matrix keeping the cells attached to the plate	-Dissociate and replate the cultures onto fresh well plates. Note that this will dilute out the CHX10+ cells.
5A(ix)	Low CHX10 percentage in human differentiations	- Initial seeding density is inappropriate - Incompatible Matrigel lot	- May need to test different lots of Matrigel to see what supports a neuronal differentiation. - Test a range of seeding densities for each cell line.
5B(i)	Cell layer peeling during staining	Cell layers are releasing during fixation and wash steps	Leave 500 μL of media in the wells and add 500 μL of 4% PFA into the wells to fix. Fix for 1 hour instead of 30 minutes. During wash steps, a transfer pipette may be used to carefully exchange liquids.
5B(vii)	Speckled signal in immunostaining	Excess secondary antibodies aggregate and do not wash out	Filter the solution with the secondary antibody to remove any aggregates. If the target has low abundance, try a lower concentration of secondary antibody (< 1:200).
Box 2: 6	Low cell survival post-thaw	Cell death during thaw	Do not let the vial of cells completely thaw in the water bath.
Box 2: 8	Low cell survival post-thaw	Excess cell debris after thaw	24 hours after thaw carefully aspirate the media and wash with 0.5 ml PBS. Aspirate PBS and add fresh NIM.