Peter van Mourik

101 Forskolin-induced swelling protocol Forskolin induced swelling (FIS) assay Timing ~5 h The FIS assay was developed to measure CFTR function in human colon organoids. Upon forskolin (fsk) stimulation (which indirectly activates CFTR through cyclic adenosine monophosphate (cAMP)), CFTR function can be assessed by observing the subsequent swelling of human colon organoids. Organoid swelling occurs through chloride excretion into the organoid lumen and osmosis. The assay can determine residual CFTR function and the effect of CFTR modulators. CRITICAL: Pre-warm 24- and 96-wells plates used for organoid culturing to 37 °C for a minimum of >1 day. This is essential to ensure that after the colon organoids are plated, the matrigel polymerizes in the well efficiently and forms stable drops. A. Preparations 1. Incubate 96-well plates at 37 °C > 7 days. It is important to have warm plates to prevent dislodging of matrigel drops. 2. Prepare cold Ad-DF+++. 3. Thaw (and keep) matrigel on ice and dilute 1:1 with cold CM +/+ (= 50 % matrigel). 4. Prepare warm CM +/ +. B. Plating human colon organoids (day 1) Note: Some CFTR modulators (e.g. correctors) require longer periods incubation. Example: correctors VX-809 and VX-661 require 24 h incubation and are added directly to the CM +/+ on the day of the organoid plating, whereas potentiator (e.g. VX-770) should be added directly before the FIS measurement. Take this into account when CM +/+ is added. See ‘ European FIS validation & standardization protocol’ for corrector preparations. 1. Perform step 1-9 of ‘General handling and passaging of human colon organoid cultures’, part B. 2. Resuspend the organoid pellet in 50% matrigel (plating volume = 4 µL * amount of wells + 50 µL) o With good quality organoid cultures, one full well of organoids from a 24- wells plate can be seeded into 10 - 20 wells of a 96-wells plate Example: 8 wells of a 7-day, good quality organoid culture can be split and seeded into ~80-160 wells of a 96-wells flat bottom plate. To avoid too sparsely seeded wells, it is advisable to first resuspend in a matrigel volume sufficient to seed 80 wells. 4