Table 4.

Details of colon seeding medium composition and preparation

Component	Stock concentration	Volume added	Final concentration	Recommended storage condition
WRN-conditioned media	100×	6.5 ml	65×	−20 °C freezer
Base media*	100×	3.2 ml*	32×	4 °C fridge
B27	50×	200 μl	1×	−20 °C freezer
N2	100×	100 μl	1×	−20 °C freezer
N-acetyl cysteine	500 mM	10 μl	500 μM	−20 °C freezer
A83-01	500 μM	10 μl	500 nM	−20 °C freezer
Thiazovivin**	6.25 mM	4 μl	2.5 μM	−80 °C freezer
SB202190	30 mM	3.33 μl	10 μM	−20 °C freezer
EGF	250 μg/ml	2 μl	50 ng/ml	−20 °C freezer
human [Leu15]-Gastrin	100 μM	1 μl	10 nM	−20 °C freezer
Prostaglandin E2	10 mM	2 μl***	5 nM	−80 °C freezer
Final volume		10.03 ml

^*Base media consists of Advanced DMEM/F12, 2mM Glutamax, 10 mM HEPES, 100 units/ml penicillin/streptomycin as described in Table 1.

^**Nicotinamide is removed and thiazovivin is used instead of Y-27632 dihydrochloride in seeding medium to allow differentiation into secretory cell types in monolayers as reported by Kozuka et al.²⁷

^***Prostaglandin E2 should be diluted before use. Make a dilution of 1 μl PGE stock to 349 pl base media. Add 2 μl PGE dilution for 10 ml media