2.8. Fsk-Induced Swelling (FIS) Assay of Intestinal Organoids

Crypt isolation from rectal biopsies, culture of organoids (F508del/F508del genotype), and FIS assay was performed as described [27]. Twenty-four hours after seeding and treatment with MCG1516A and VX-661, organoids were acutely stimulated (30 min) with Fsk (0.02–5 µM) plus VX-770 (3 µM) and live-cell imaging was performed using a confocal microscopy (Leica TCS SP8) with a 5× objective for 60 min at 37 °C. FIS was quantified using the area under the curve (AUC, t = 60 min, baseline = 100%) and a CellProfiler-based algorithm.