Peter van Mourik

89 Forskolin-induced swelling protocol 5. Check the crypt density by plating a test droplet and view under the light microscope. Preferably do not plate out crypts too dense to allow for efficient (out)growth and proliferation of the structures. The ideal density is 15- 20 crypts per 7.5 µL of matrigel drop. Note: the crypt density can be adjusted according to the observations in the test droplet. Too dense: If crypts are too densely seeded, dilute the sample in more matrigel and seed additional wells with the extra matrigel. Too sparse: If crypts are too sparsely seeded, crypts can be centrifuged at 130 g 5 min at 4 °C and resuspended in smaller volumes of matrigel to increase the density. Note: Prevent bubble formation during pipetting and plating. 6. Plate 4* 7.5 µL of matrigel with about 15-20 crypts per drop of a (pre-warmed) 24-wells plate. 7. Resuspend the matrigel after plating out 3 wells to ensure the crypts have not settled at the bottom of the microcentrifuge tube. 8. Incubate 30 min. at 37 °C. 9. Add 500 µL of pre-warmed CM +/+ with additional gentamicin (50 µg/mL) and vancomycin (50 µg/mL). An example of crypts directly after isolation can be found in Figure 5. Note: gentamicin and vancomycin are only added to CM +/+ in the first week of culturing 10. Incubate for 7 days at 37 °C, 5 % CO 2 . Figure 5. Crypts on day 0 directly after isolation (bright field, 4 x magnification). 4