Table 1: Appropriate volumes of papain, albumin-ovomucoid, and final suspension (Hibernate E) used in dissociation steps.

The appropriate volumes of papain and albumin-ovomucoid to be used with various numbers of ventral midbrain and ventral spinal cord tissues were modified from the manufacturer’s instruction after several rounds of optimization. Since tissues are subject to stress during dissociation and sorting, pooled collection of more than 10 ventral midbrains and more than 3 ventral spinal cords is recommended. The volume of papain was determined by considering the balance between effective dissociation and the stress of this procedure. The volume of albumin-ovomucoid inhibitor solution is half of that of papain. The appropriate volume of Hibernate E final suspension was determined such that cell density does not exceed 10⁷ cells/ml, but also such that the cells do not become excessively diluted.

Number of midbrains (X)	Papain	Albumin-ovomucoid	Hibernate E
10≦X≦20	200 μl	100 μl	600 μl
20<X≦30	300 μl	150 μl	700 μl
30<X≦40	400 μl	200 μl	800 μl
Number of spinal cords (Y)	Papain	Albumin-ovomucoid	Hibernate E
3≦Y≦5	200 μl	100 μl	500 μl
5<Y≦10	400 μl	200 μl	800 μl
10<Y≦15	600 μl	300 μl	1200 μl