MATERIALS AND METHODS Mice. Mice with gene-targeted disruption selleck chemicals llc of the murine homolog of Cftr [Abcc7, Cftr knockout (KO)] and wild-type (WT) littermates were used. All comparisons were made with sex- and age-matched (+/+ or +/?) siblings. The mutant mice were identified using a PCR-based analysis of tail snip DNA, as previously described (9). All mice were maintained ad libitum on standard laboratory chow (Formulab 5008, Rodent Chow; Ralston Purina) and distilled water containing Colyte laxative to avoid intestinal obstruction in the Cftr KO mice (9). Mice were housed individually in a temperature (22�C26��C)- and light (12:12-h light-dark cycle)-controlled room in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the Dalton Cardiovascular Research Center, University of Missouri.

All experiments involving animals were approved by the University of Missouri Animal Care and Use Committee. Enteroid culture. The enteroid culture method was modified from Sato et al. (52). Mouse proximal small intestine (~10 cm) was excised, opened longitudinally, and washed with ice-cold PBS. The intestine was cut into small pieces (~4- to 5-mm diameter) and incubated in ice-cold PBS containing 2 mM EDTA for 30 min. After being rinsed once with ice-cold PBS to remove EDTA, the intestinal fragments were resuspended four times in ice-cold PBS (10 ml) by repeated, vigorous pipetting using a 10-ml pipette. After each resuspension, the tissue fragments were allowed to settle and the supernatant from the first two resuspensions was discarded.

The supernatant from the last two resuspensions was collected and passed through a 70-��m cell strainer (Becton-Dickinson Bioscience, Franklin Lakes, NJ) to remove tissue fragments. Crypts in the strained solution were separated from suspended single cells by centrifugation (200 g, 1 min). The crypt pellet was resuspended with cold PBS and mixed 1:2.5 vol/vol with Matrigel (BD Bioscience) for plating (e.g., ~100 ��l/35-mm petri dish). After polymerization of the Matrigel, culture medium composed of Ham’s F-12 containing 5% FBS, 50 ��g/ml gentamicin, 125 ng/ml Rspondin1, 25 ng/ml noggin, and 12.5 ng/ml epidermal growth factor (EGF) was added and changed every 2�C4 days. The percentage of FBS and concentrations of Rspondin1, noggin, and EGF are 25% of those used in the method by Sato et al.

(52). Preliminary studies of WT enteroids grown in increasing dilutions of these growth factors indicated that enteroid development, as assessed by size and crypt numbers, was slowed by ~1�C2 days but not prevented using 25% of the original concentration (data not shown). For enteroid passage at 7�C10 days postplating, culture dishes were rinsed twice with ice-cold Entinostat PBS before addition of cell recovery solution (BD Biosciences).