Human enteroids recapitulate two major functions of the small intestine: Na+ absorption and Cl- secretion. (a) Enteroids exhibit NHE3 activity, which can be inhibited by forskolin (FSK) treatment before or after initiation of transport measurements. Differentiated enteroids were loaded with the pH-sensitive dye SNARF-4F and prepulsed with 50 mM NH4Cl to acidify the cytosol. Na+-dependent alkalinization in the presence of 50 μM HOE694 is due to NHE3 activity (no alkalinization in the presence of the NHE3 inhibitor S3226, added after HOE694). n = 6 for each condition. CTL, control; STD, pH standards; TMA, tetramethylammonium chloride. (b) Duodenal enteroid lumens (L) significantly dilate in response to elevated cAMP levels via FSK treatment. After FSK treatment, the size of enteroid lumens increased 203 ± 16% over controls. The same optical section of enteroids loaded with SNARF-4F was compared before (red) and after (green) 30 minutes of FSK treatment. Arrows indicate change in position of epithelial layer due to luminal dilation. n = 3 for each experiment.