Peter van Mourik

92 Chapter 4 Figure 7. Put a p200 tip on top of a p1000 filter tip to disrupt the organoid structures. Note: steps 5 and 6 are relevant when colon organoid cultures are passaged before performing a FIS experiment or to clean an organoid culture that has many differentiated structures which need to be removed (see figure 10C). Otherwise proceed with step 7. 5. Add 5 mL of cold Ad-DF+++ to the 1 mL of disrupted organoids. 6. Clean up the organoid suspension by resuspending the organoids and let the bigger, differentiated organoid structures sink down in a tube angles at 70° for 10 s (see figure 8). Take up the smaller organoids by removing 1 mL from the top of the medium and transfer this to a sterile 15 mL tube on ice. Repeat this step until only 0.5 - 1 mL medium with bigger structures is left. Note: if many large structures are still left in the remaining 1 mL after steps 5 and 6 (visual by eye), repeat steps 4-6 but during step 4 only disrupt 10 times.