Peter van Mourik

106 Chapter 4 0.25 mg R-spo3 Stock and aliquot volume dependent on LOT#. Store for 3 months at -80 °C. 35 mL 50% Matrigel Aliquot 2 x 10 mL vials in 500 µL per microcentrifuge tube and store at -20 °C B. General experimental set up The FIS validation assay is based on the experimental set up as previously performed and described (Dekkers et al., 2016, Science). 1. Each reference organoid line was tested using four different conditions (basal CFTR function, VX-661; VX- 770 and VX-661 + VX-770, each in combination with 8 different fsk concentrations (0.008; 0.02; 0.05; 0.128; 0.32; 0.8; 2 or 5 µM) which results in 32 different experimental conditions. 2. Biological replicates for each condition were measured in duplicate and experiments were repeated n=3. 3. Human colon organoids were plated in a 96-well plate as schematically depicted in table 6. 4. VX-661 was added 24 h before the FIS experiment 5. VX-770 was added acutely before the FIS experiment was initiated. Table 6. Experimental plate set up for measurement of drug response of reference organoid cell lines for validation data. (Day 1) CM +/+ CM +/ + + VX-661 3 µM CM +/+ CM +/ + + VX-661 3 µM (Day 2) Fsk stimuli Fsk stimuli + VX-770 (3 µM) Fsk concentration 0.008 µM 5 µM 0.008 µM 5 µM 0.008 µM 5 µM 0.008 µM 5 µM 0.008 µM 5 µM 0.008 µM 5 µM 0.008 µM 5 µM 0.008 µM 5 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.02 µM 2 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.05 µM 0.8 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM 0.128 µM 0.32 µM Per organoid reference line