Conflicts with diarrheal pathogens trigger rapid evolution of an intestinal signaling axis

Clayton M. Carey; Sarah E. Apple; Zoe A. Hilbert; Michael S. Kay; Nels C. Elde

Organoid swelling assay Organoids were grown in a 24 well plate for 5-6 days after passaging prior to swelling assays at a concentration of 100-500 organoids per well. Organoids were imaged on an ImageXPress Pico Automated Cell Imaging System (Molecular Devices) using the live cell imaging cassette with temperature set to 37˚C, 5% CO2, and humidity levels 85-95%. These parameters were monitored and remained constant throughout each experiment. For swelling analysis, ST toxins were added directly to the organoid culture media to a final concentration of 5 µM. Following addition of toxin, a 24 mm 2 area of each well (corresponding to ~13% of the total well area) was imaged every 10 min at 4x magnification for the full 2 hours of the experiment. The imaged area was constant across the experiment and analogous between wells and contained at least 50 organoids for each analyzed strain. 20 stacked transmitted light images were collected for each well with a focus step of 25 µm and the Best Plane image was calculated by the ImageXPress software. GFP images were collected at the beginning and end of the experiment using the same imaging parameters, and both the best focus and maximum fluorescence images for each stack were calculated and output by the ImageXPress software.