Due to selleckchem the hydrophobic nature of CFTRinh172, a longer incubation for diffusion is required for enteroids embedded within the Matrigel and preliminary studies indicated that a 1-h period provided optimal inhibition of forskolin-induced WT crypt cell shrinkage (data not shown). Cell height, i.e., distance from the apical to basolateral membrane, and the mean height (h) and width (2r) of the crypt and its corresponding crypt lumen in the midcrypt region (+8 to +15 cell position) were measured before and 10 min after treatment from captured images using ImagePro Plus 6.3 software (Media Cybernetics, Bethesda, MD). The percent change in cell height was calculated by dividing the change in cell height by the basal (pretreatment) cell height * 100.

The percent change in crypt epithelial volume was calculated by dividing the change in crypt epithelial volume by the basal (pretreatment) crypt epithelial volume *100. Crypt epithelial volume at both points was calculated, assuming a cylindrical shape of the crypt and crypt lumen, by subtracting the crypt lumen volume from the total crypt volume, using the formula for a cylinder volume ��?h?r2. Stable, morphological landmarks were used to determine the cylinder height at locations within the +8 to +15 cell position above the crypt base before and after treatments. Cell height and epithelial volume at the crypt base were not determined because nearly complete Paneth cell degranulation during some treatments, e.g., carbachol, confounded accurate measurement of the parameters. Confocal microscopy measurement of intracellular pH.

Enteroids were cultured for 4�C5 days on glass-bottomed 35-mm Fluorodishes (World Precision Instruments), as described in Enteroid culture. Enteroid epithelium was loaded with 40 ��M ratiometric fluorescent pH indicator SNARF-5F (Invitrogen) and 2.5 ��M quinicrine (Sigma-Aldrich, St. Louis, MO) in culture medium for 30 min at 37��C. Enteroid cultures were mounted on the stage of a TCS SP5 Confocal-Multiphoton microscope built on a DMI6000 inverted platform (Leica) and fitted with a temperature-controlled incubator containing a 95% O2-5% CO2 atmosphere (Life Imaging Services, Basel, Switzerland). Organoids were continuously superfused with KBR + TES (pH 7.4, gassed with 95% O2-5% CO2, 37��C). The Dacomitinib excitation source for quinicrine was a 488-nm argon laser, and images were collected at an emission wavelength of 500�C540 nm. The excitation source for SNARF 5F was a 514 nm argon laser and images were collected at dual emission wavelengths (580 �� 30 and 640 �� 30 nm). Z stacks (~30 1-��m slices) of individual crypts were acquired using the two sets of excitation-emission wavelengths (avoiding optical contamination of SNARF 5F fluorescence by quinicrine).