Table 2D.15.1.

Troubleshooting Guide for Common Problems During ESC Differentiation and Viral Tracing

Problem	Possible cause	Solution
Poor ESC cell growth on gelatin-coated plates	First feeder-free passage on gelatin-coated plates Bottom of culture dish is not covered with gelatin Cells are differentiating	Passage at least three times and evaluate cell growth Ensure that gelatin-water covers bottom of dish during dish preparation Add sufficient LIF to media
Neurospheres attach to plate	Bacteriological petri dishes promote attachment Density of cells in suspension is too high	Obtain petri dishes from suggested manufacturer Start suspension culture with suggested number of dissociated ESCs
ESCs do not differentiate into neuronal lineage with high yield	Decreased pluripotent marker expression in starting ESC culture Retinoic acid is of insufficient quality Retinoic acid was omitted	Obtain a new batch of ESCs with high pluripotent marker expression Obtain a new batch of retinoic acid Restart suspension culture
ESC-derived neurons do not express GFP on RV infection	ESCs do not express sufficient amounts of fluorescent reporter, rabies-G, and TVA receptor	Verify expression via fluorescent reporter expression, ESC viral infection, and immunoblotting
Transsynaptic tracing is not observed	ESC-derived neurons do not make sufficient connections with host neurons	Allow a minimum of 5 days for ESC-derived neurons to form connections with primary neurons
Primary neuronal culture dies upon RV infection	RV is allowed to spread longer than 14 days and kills cells	Perform observation/experimentation as early as 5–7 days post RV infection but no longer than 10 days