Table 1.

Summary of Hypothalamic Neurosphere Culturing Protocol

Culture Media	Insulin-supplemented media (ISM) provides required environment for strong growth without introducing exogenous hormones. Ingredients: 1x DMEM/F12 100 U/mL penicillin-streptomycin, 1 mg/mL heparan sulfate sodium salt 0.6% glucose 0.1125% bicarbonate 0.005M HEPES 2mM glutamine 20 μg/mL apo-transferrin 2.5 μg/mL insulin 1 μg/mL putrescine dihydrochloride 3mM sodium selenite
Clonal Density	Primary cells: 5000 cells/well Secondary cells: 250 cells/well (each in 1mL volume in standard 24-well plate)
Refreshment Routine	Refresh 50% media and treatment components at 5DIV
Growth Factors	ISM—Require both EGF & bFGF (2ng/mL or 20ng/mL)
Shh Supplement	Recommended to enhance neurosphere growth (2ng/mL)
Fungizone Supplement	Not recommended, strongly inhibits neurosphere growth