Peter van Mourik

109 Forskolin-induced swelling protocol Table 7. Workflow and timelines of FIS assay validation FIS ASSAY VALIDATION - WORKFLOW & TIMELINES (SUGGESTED) Wk nr Action Extra comments > Wk 0 Prepare all media prior to starting the reference organoid cultures. Wk 0 Thaw 1 cryovial into 3- 4 wells (pre-warmed 24-well plate) per reference organoid line. ● Check the seeding density under the microscope. ● Add ROCKI (10 µM) to CM +/+. Wk 1 If budding organoid structures are visible: ● Switch to regular CM +/+. → Disruption 1-2 full well and seed into 4 fresh wells ● Skip organoid clean up step. If organoids are still very small: → Take up all 4 wells, centrifuge and seed into 4 fresh wells Wk 2 If budding organoid structures are visible: ● Skip organoid clean up step during passaging. → Passage 1-2 full wells of organoids into 4 new wells → Pool organoids from 2 wells and freeze a minimum 2 cryovials WCB If organoids are still very small: → Take up all 4 wells, centrifuge and seed into 4 new wells Wk 3 All colon organoid structures should be budding, proliferating structures (Ch. 7, pic 6): ● Skip organoid clean up step during passaging. → Passage 1-2 full wells of organoids into 4 new wells → Pool organoids from 2 wells and freeze a minimum 2 cryovials WCB Wk 4 Take up 3 - 4 full wells: → Follow the organoid disruption & clean up steps. → Seed 8 wells per reference line for FIS assay experiment n=1 in wk 5. Wk 5 Take up 8 full wells: ● Check if confocal settings and image analysis are optimal. 4