Organoid culture media

Reagent	Final concentration	Amount
DMEM/F12 advance	1×	47 mL
HEPES (1 M)	10 mM	500 μL
Penicillin-Streptomycin (10,000 U/mL)	100 U/mL	500 μL
GlutaMAX (100×)	1×	500 μL
B27 (50×)	1×	1 mL
SB431542 (10 mM)	0.5 μΜ	2.5 μL
mFGF10 (100 μg/mL)	100 ng/mL	50 μL
hEGF (500 μg/mL)	0.1 μg/mL	10 μL
hNoggin (200 μg/mL)	100 ng/mL	25 μL
hR-spondin1 (600 μg/mL)	0.6 μg/mL	50 μL
mWNT3A (100 μg/mL)	100 ng/mL	50 μL
Total	n/a	50 mL

Organoid media can be stored in 1 mL aliquots at −80°C. Once thawed, use immediately and do not refreeze.