All enteroid media were prepared as reported previously (32 (link)). Advanced Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium supplemented with 1× GlutaMAX (Gibco), 10 mM HEPES (Quality Biologicals), and 100 Units/ml penicillin-streptomycin (Gibco) was used as the basal medium.
Complete medium with growth factor (CMGF+) is comprised of 22.3% (vol/vol) basal medium, 50% (vol/vol) Wnt3a-conditioned medium, 15% (vol/vol) R-spondin-1-conditioned medium, 10% (vol/vol) noggin-conditioned medium, 1× B27 supplement (Gibco), 1 mM N-acetylcysteine (Sigma), 1× Primocin (InvivoGen), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10 μM SB202190 (Tocris).
Differentiation medium is comprised of 86.2% (vol/vol) basal medium, 10% (vol/vol) noggin-conditioned medium, 1 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), and 500 nM A83-01 (Tocris).
Complete medium with growth factor (CMGF+) is comprised of 22.3% (vol/vol) basal medium, 50% (vol/vol) Wnt3a-conditioned medium, 15% (vol/vol) R-spondin-1-conditioned medium, 10% (vol/vol) noggin-conditioned medium, 1× B27 supplement (Gibco), 1 mM N-acetylcysteine (Sigma), 1× Primocin (InvivoGen), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10 μM SB202190 (Tocris).
Differentiation medium is comprised of 86.2% (vol/vol) basal medium, 10% (vol/vol) noggin-conditioned medium, 1 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), and 500 nM A83-01 (Tocris).
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