Peter van Mourik

93 Forskolin-induced swelling protocol Figure 8. Hold the 15 mL tube tilted at 70 ° and transfer the organoids from the top of the medium to a new tube. 7. Top up the 15 mL tube containing the smaller, disrupted organoids with 10 mL cold Ad-DF+++. 8. Centrifuge at 130 g for 5 min at 4 °C. 9. Aspirate the supernatant and keep the organoid pellet. CRITICAL: the colon organoid pellet is vulnerable and can be easily lost through aspiration of the supernatant. Be careful while aspirating the media: it is best to leave > 50 µL of supernatant on the organoids to avoid pellet aspiration. Remove the last supernatant with a p200 tip. C. Plating colon organoids in 24-well plates 1. Resuspend the organoid pellet in an appropriate volume of 50% matrigel. The volume of matrigel needed is 30 µL per seeded well of a 24-wells plate assuming enough organoids have been passaged. Example: 2 wells of a 7-day old, good quality organoid culture can be split into approximately 8 - 16 new wells of a 24-wells plate. To avoid too sparsely seeded wells, it is advisable to first resuspend in a matrigel volume sufficient to seed 6 wells. Therefore, the pellet can be resuspended in approximately 180 µL of 50% matrigel. In case the organoids are too densely seeded, the organoid suspension can be diluted by adding more matrigel. 2. Check the number of organoids in a 10 ml test drop of matrigel by light microscopy, to decide if the organoid suspension should be further diluted (the 4