Peter van Mourik

113 Forskolin-induced swelling protocol 1. Percent increase of organoid area per fsk concentration and condition. 2. The area under the curve (AUC) of the percent increase of organoid area per fsk concentration for all conditions. Calculations from raw analysis FIS data in Excel Exported data from imaging software is further processed and calculated in Excel. Data is transposed and calculated in a pre-set format according to the linear trapezoidal method that calculates the average increase over a given time window that is multiplied by the time units within the interval. First, the relative increase over time in area µm 2 per well derived from the exported data is normalized per well to time point 0. Time point 0 is set to 100% and time point 10 – 60 min are normalized accordingly (see table 4, example for 8 measured wells with 4 different conditions; time clips are presented as columns). The relative increase in swelling per well is now expressed in percentages creating 7 data points per well, which can be expressed to show swelling increase per well in time. To more condensely express swelling data (e.g. various forskolin concentrations) in one graph, the area under the curve (AUC) of the relative increase of swelling per well over 60 min can be calculated to create a single data point per well. By using the AUC values, the large datasets can be condensed into graphs capturing all data (see example data in table 4 & Figure 13). Figure 13. (A) Raw data (area µm2 per well per time point) can be normalized to time point 0 min. Nor- malized data depicted in a graph shows relative increase in swelling per well over time (baseline is set to 100%). Here, duplicate wells are measured for swelling at 0.02; 0.128; 0.8 and 5 µM fsk for 60 min. (B) The AUC of relative increase in swelling is calculated in Excel. (C) Using AUC calculations for the different conditions, a condensed graph can be created which represents overall relative swelling data per condition over 60 min. Graph C shows the average AUC data of the duplicate wells (from B) with SD. 4