The 580-to-640-nm ratio was converted to pHi using a standard curve generated by K+/nigericin technique (56). Materials. SNARF 5F acetoxymethyl ester was obtained from Invitrogen. Forskolin and Cftrinh172 were obtained from Enzo Life Sciences (Farmingdale, NY). EGF and noggin were obtained from R&D Systems (Minneapolis, Ganetespib cancer MN). Recombinant Rspondin1 was isolated as described previously (43). All other materials were of analytical grade and obtained from either Sigma-Aldrich or Fisher Scientific. Statistics. All values are reported as means �� SE. Data between two groups were compared using a two-tailed Student t-test assuming equal variances between groups. Data from multiple treatment groups were compared using a one-way ANOVA with a post hoc Tukey’s t-test. A probability value of P < 0.

05 was considered statistically significant. RESULTS The enteroid model. With the use of modifications of Sato et al. (52; see Enteroid culture), freshly isolated crypts initiated a process of crypt fission within 1�C2 days to form an organoid with multiple crypts and a central, epithelial-lined cavity containing sloughed apoptotic cells (Fig. 1A). As shown in Fig. 1, B and C, goblet and Paneth cells were well differentiated and readily identified morphologically by light microscopy based on the characteristics of granule size, theca position, and location within the crypt, as described previously (8, 59). Goblet cell numbers in the WT enteroid crypts approximated WT small intestine crypts (WT enteroid: 4.8 �� 0.2 goblet cells/crypt cross section; WT small intestine: 5.2 �� 0.

3 goblet cells/crypt cross section, NS, n = 6�C7 mice). Paneth cell numbers were not estimated due to the difficulty of distinguishing individual cell borders in the histological sections. Goblet and Paneth cell functional activity was demonstrated by induction of degranulation upon basolateral exposure to the muscarinic agonist carbachol (100 ��M; Fig. 1, B and C). Fig. 1. Enteroid model. A: development over 8 days of an enteroid structure from a single, freshly isolated murine crypt in Matrigel suspension culture (magnification: ��10 and ��20). B: goblet cell in enteroid crypt (left) and time course of goblet … Rapid development of the enteroids raised the question of whether the epithelial proliferation rate of the enteroids recapitulates that of crypts in vivo.

To estimate crypt epithelial proliferation rate in vivo, EdU labeling was used to identify S-phase cells in crypt cross sections of duodenal sections from WT mice (1 h post-EdU injection ip). As shown by the histogram in Fig. 2A, the number of EdU+ cells per crypt exhibited a wide range of proliferation, varying from quiescent Batimastat crypts (0 EdU+ cells) to highly proliferative crypts (26 EdU+ cells). The number of EdU+ cells per crypt cross section was normally distributed with the means = 10.4 �� 2.2 EdU+ cells/crypt.