Peter van Mourik

104 Chapter 4 9. Measurement settings for 60 minute measurement: o Interval = 10 min, o Cycles = 7 (Cycle 1 is t = 0). Note: the acquisition of images from the total 96-well plate in each time point should not exceed 5 min to ensure optimal comparison of time points between wells. 10. Make sure the calcein green signal in the organoids structures is slightly oversaturated and the signal to noise ratio is optimized as much as possible (this is essential for optimal recognition of the organoid structures by the imaging software). 11. Add 50 µL Ad-DF+++ containing fsk stimuli (and/or CFTR-modulators) to each well containing 50 µL of CM +/+. It is preferred to add the stimuli from a 96-well dummy plate with an 8-multichannel for accuracy and efficiency if the size of the live cell chamber allows this. If this is not possible, add the stimuli with a regular pipet. 12. Start the acquisition immediately after stimuli are added. Figure 12. Example of organoid swelling after 60 min FIS assay of F508del/ F508del calcein green stained colon organoids in the absence or presence of VX-661 (3 µM) & VX-770 (3 µM) and fsk (0.8 µM).