Peter van Mourik

111 Forskolin-induced swelling protocol D. Preparation of CFTR corrector VX-661 (day 1) 1. Prepare CM +/+ with VX-661 before plating out the organoids. VX-661 needs to be added 24 h in advance of the FIS measurement in CM+/+ with a final concentration of 3 µM. For example: from a 20 mM stock: add 95 µL of Ad- DF+++ to 5 µL of 20 mM stock creating 1 mM of VX-661 solution. 2. Per 96-well plate add 9.6 µL of 1 mM VX-661 to 3 mL of CM+/+ creating a final concentration of 3.2 µM. 3. Add 50 µL to each well that requires 24 h of VX-661 incubation (see table 6 for plate lay out) 4. The rest of the wells can be filled with regular CM +/+. E. Preparation of CFTR stimuli VX-770 & fsk (day 2) Per 96-well plate: 5. Add 1 µL fsk (10 mM) to 1 mL Ad-DF+++ (= fsk (10 µM)), final concentration in assay will be 5 µM). 6. Add 2 µL of VX-770 stock (20 mM) to 6 mL of Ad-DF+++ (= VX-770 6.4 µM, final concentration will be 3.2 µM. 7. Take 1 mL of VX-770 6.4 µM solution and add 1 µL fsk (10 mM) (= fsk + VX-770 solution) 8. Prepare the titrations (serial dilution) according to table 8 below. Resuspend every suspension 10 times and use a new tip for every dilution step. Table 8. Preparation of the fsk titration with and without VX-770 added directly before the start of the FIS assay. Order of addition to plate Final conc. (µM) Ad-DF+++ (µL) Fsk titration (column 1-4) Ad-DF+++ (µL) + VX770 3 µM Fsk titration in 3 µM VX-770 (column 5-8) 8 5 - 1000 µL fsk (10 µM) - 1000 µL fsk / VX-770 mix 7 2 600 µL Ad- DF+++ 400 µL from 8 600 µL Ad- DF+++-770 400 µL fsk / VX-770 mix from 8 6 0.8 600 µL Ad- DF+++ 400 µL from 7 600 µL Ad- DF+++-770 400 µL from 7 5 0.32 600 µL Ad- DF+++ 400 µL from 6 600 µL Ad- DF+++-770 400 µL from 6 4 0.128 600 µL Ad- DF+++ 400 µL from 5 600 µL Ad- DF+++-770 400 µL from 5 3 0.05 600 µL Ad- DF+++ 400 µL from 4 600 µL Ad- DF+++-770 400 µL from 4 2 0.02 600 µL Ad- DF+++ 400 µL from 3 600 µL Ad- DF+++-770 400 µL from 3 1 0.008 600 µL Ad- DF+++ 400 µL from 2 600 µL Ad- DF+++-770 400 µL from 2 4