Peter van Mourik

79 Forskolin-induced swelling protocol Preparing 2x colon organoid medium -/- Timing ~1 h Table 2 shows how to prepare 2x colon organoid medium (2x CM) -/- by addition of all reagents except WCM and R-spo3. It is advisable to calculate the medium amounts that will be used within the following 2.5 months and adjust the volume of 2x CM -/- preparation. Table 2: Preparation of 2x colon organoid medium (CM) -/-. Reagent Final conc. 100 mL total medium (50 mL 2x CM -/-) Ad-DF+++ 36.8 % 36.8 mL NCM 10 % 10 mL B27 2 % 2 mL N-acetylcysteine 1.25 mM 250 µL hEGF 50 ng/mL 10 µL Nicotinamide 10 mM 1 mL A83-01 500 nM 10 µL P38 inhibitor 10 µM 33 µL Gastrin 100 µM 5 µL Primocin 100 µg/mL 0.2 mL Store 2x CM -/- in aliquots at – 20 °C for a maximum of 2.5 months. For example, aliquot 50 mL in 100 mL bottles so WCM can be added when preparing CM +/+ as described in the following section. Human colon organoid medium +/+ preparation Timing ~1 h When preparing fresh CM +/+ for direct use in human organoid cultures, thaw a bottle of frozen 2x CM -/- over 8-12 hours at 4 °C (preferably in the fridge) and add WCM and R-spo3 the following day. Table 3 shows the ratio of adding WCM and R-spo3 to create 100 mL total volume of CM +/+. CM +/+ can be used for 10 days if stored at 4 °C. Table 3. Preparation of colon organoid medium (CM) +/+ Reagent Final conc. 100 mL CM +/+ Storage 2x CM -/ - 50 % 50 mL 10 days at 4 °C WCM 50 % 50mL 2 months at 4 °C R-spo3 250 ng/mL Depends on LOT# 7 days at 4 °C 4