Peter van Mourik

103 Forskolin-induced swelling protocol Note: Regular organoid passaging and organoid seeding for FIS experiments can be combined. Guideline: prepare excess organoid suspension for seeding in a 96- well plate. Remaining organoid suspension can be diluted at least 1:1 and used for seeding in a 24-well plate for a following 7-day culture. C. FIS assay & confocal microscopy (day 2) 1. Incubate Ad-DF+++ at 18 – 37 °C. 2. Turn on the live cell compartment of the confocal microscope and pre-incubate at 37 °C and 5% CO 2 (Pre-heating the live cell compartment takes a minimum of 30-45 min). 3. Prepare fsk and/or CFTR-modulators as desired. Prepare fsk and CFTR- modulator compounds in a 2 x concentration in Ad-DF+++ so when 50 µL is added to the organoids in a well of the 96-well plate containing already 50 μL of CM+/+, , the 1:1 dilution creates a 1 x final concentration of the desired fsk or compound concentration. Example: if the desired final concentration of fsk within the well is 5 µM, prepare a fsk dilution of 10 µM. If 50 µL of 10 µM fsk is then added to the 50 µL CM +/+, the final concentration will be 5 µM. 4. Prepare 8.4 mM stock of calcein green AM by adding 6 µL of DMSO to one 50 µg calcein green vial. For one full 96 well plate, add 1 µL of 8.4 mM calcein green solution in 1000 µL Ad-DF+++. 5. Add 5 µL of calcein green solution to each well (final concentration is 0.84 µM) 6. Gently resuspend the well 2-3 times with a multichannel for homogeneous staining and efficient uptake of calcein green by the organoid structures. Try tilting the plate and point tips of multichannel in the corner of each well to prevent touching the matrigel drop. 7. Incubate the plate at 37 °C, CO 2 5 % for 15 - 30 min before starting the experiment. 8. Put the 96-well plate in the plate holder of the live cell imaging device and ensure the plate is in fixed position. Live cell imaging settings: o Organoid structures with calcein green stain can be visualized upon emission at 488 nm and excitation at 515 nm (detection wavelength range 450 – 700 nm). o Use 5 x objectives and ensure an overview of the full matrigel drop by adjusting the focus. o Set the position (x, y) and focus (z) of the matrigel drops in the acquisition software. Note: an autofocus option may be used when available. 4