Peter van Mourik

69 Forskolin-induced swelling protocol Before you begin: CRITICAL: All laboratory procedures related to colon organoid or cell cultures should be performed in a laminar flow to avoid contaminations and to protect the operator from biological risks. A. Preparations 1. Prepare Dulbecco’s Modified Eagles Medium with additional 1 % (v/v) pen- strep (10,000 U/mL) and 10 % (v/v) FBS (DMEM +/+) and pre=warm to 37° C B. Wnt-3A conditioned medium (WCM) production Note: it is strongly advised to produce and mix multiple batches (2-3) of WCM to compensate for batch-batch variation during WCM production. Each round can produce around 1100 mL of WCM leading to a WCM pool of 2200 (mix of 2 batches)– 3300 mL (mix of 3 batches) of which the total production time is between 20 (two batches) and 24 (3 batches) days. 1. Thaw a cryovial with L-Wnt-3A cells by putting 13 mL of pre-warmed DMEM +/+ medium in a 15 mL tube (when thawing multiple cryovials, use a 50 mL tube with 30 mL DMEM +/+). 2. Transfer the cells from the cryovial to the 15 mL tube by resuspending the cells in DMEM +/+. 3. Centrifuge the 15 mL tube at 450 g for 5 min. Remove the supernatant and resuspend the cell pellet in 25 mL pre-warmed DMEM +/+ with additional 31.25 µL zeocin (final concentration 125 µg/mL). 4. Make sure that a homogenous cell suspension is obtained and transfer the cell suspension to a T175 flask. 5. Incubate the T175 flask for 3-4 days at 37 °C, 5% CO 2 . 6. Split the cells when 90-100% confluency is achieved into 6 x T175 flasks: 7. Remove the DMEM +/+ from the T175 flask. 8. Gently wash the cells once with 5 mL PBS0 and discard the PBS0. 9. Add 1.5 mL 0.05 % Trypsin EDTA and leave at 18-23 ° C for 7 - 10 min until the cells are detached. 10. Add 10.5 mL DMEM +/+ medium, take up all the cells and resuspend until a homogenous cell suspension is achieved. 11. Add 2 mL DMEM +/+ of the cell suspension to each new T175 flask and top up to 25 - 30 mL of total DMEM +/+ per flask: > 5 x T175 flask in DMEM +/+ > 1 x T175 flask in DMEM +/+ with zeocin, use this flask for the next production batch when the cells are 90-100% confluent again (usually after 3 - 4 days). 4