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. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: Cell Stem Cell. 2022 Dec 1;29(12):1685–1702.e22. doi: 10.1016/j.stem.2022.11.004
Day of Neuralization Supplements Base Media Media changes
Day −1 THZ (1 μM) Stemflex (50%) \ KSR (50%)
Day 0 LDN (500 nm), SB431542 (10 μM) KSR (100%)
Day 1 – 2 LDN (500 nm), SB431542 (10 μM), SAG (2 μM), Purm (2 μM) KSR (100%) Every 2nd day
Day 3 LDN (500 nm), SB431542 (10 μM), SAG (2 μM), Purm (2 μM), CHIR (3 μM) KSR (100%)
Day 4 – 5 LDN (500 nm), SB431542 (10 μM), SAG (2 μM), Purm (2 μM), CHIR (3 μM) KSR (66%) \ N2B27 (33%) Every 2nd day
Day 6 LDN (500 nm), SAG (2 μM), Purm (2 μM), CHIR (3 μM) KSR (33%) \ N2B27 (66%)
Day 7 LDN (500 nm), CHIR (3 μM), FGF8 (100ng/ml) KSR (33%) \ N2B27 (66%)
Day 8 – 15 LDN (500 nm), CHIR (3 μM), FGF8 (100ng/ml)
**THZ (1 μM) 24h before and after splitting		 N2B27 (100%) Every 2nd day
Day 16 – 20 THZ (1 μM) N2B27 (100%) Every 2nd day