. Author manuscript; available in PMC: 2022 Jan 1.

Published in final edited form as: Curr Protoc. 2021 Jan;1(1):e21. doi: 10.1002/cpz1.21

Table 4.

Troubleshooting

Step	Problem	Possible reason	Potential solution
Basic Protocol 1, step 16	Little to no cell reattachment after first passage of differentiating neural crest	Cells were passaged prior to 100% confluence.	Passage one of the back-up wells retained in step 10.
Basic Protocol 1, step 16		Some hPSC lines show poor reattachment at first passage.	Include 10 μM Y-27632 during first passage.
Support protocol 1, step 11	Low % p75-NGFR⁺ HNK-1⁺ cells in pre-sort sample.	Differentiation efficiency is hPSC line-dependent.	Optimize hPSC seeding density and/or molecule concentrations in E6-CSFD medium (see recipe), especially CHIR and/or FGF2 concentration (Basic Protocol 1, step 6).
Support protocol 1, step 11	< 95% p75-NGFR⁺HNK-1⁺ cells in post-sort sample	Low % p75-NGFR⁺HNK-1⁺ cells in pre-sort sample.	See above.
		Cells were incompletely singularized prior to MACS.	Extend Accutase treatment time; filter cell suspension through 40 μm cell strainer prior to MACS. (Basic Protocol 1, steps 19 and 20).
		MACS column was overloaded or insufficiently washed.	Load ≤ 2×10⁷ cells per column. Allow MACS buffer to stop flowing before adding next 3 mL. Perform at least 3 washes (Basic Protocol 1, steps 26 and 27).
Support protocol 3, step 13a	Low % NG2⁺ cells	Timing of increase in NG2 expression is variable and may be hPSC line-dependent.	Continue to culture differentiating pericyte-like cells in E6 + 10% FBS for an additional ~3 days.