Caco-2 intestinal epithelial cells form polarized cysts in three dimensions. Caco-2 cells plated as a single-cell suspension in three dimensions were fixed and stained after 12 d. (A–C) Single confocal sections through the middle of cysts stained for DNA (blue) and actin (red; A); DNA (blue) and aPKC (red; B); and DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; C). Phase-contrast images (differential interference contrast [DIC]) are also shown. (D) Caco-2 plated as a single-cell suspension in three dimensions were cultured for 7 d and treated with 0.1 μg/ml CTX. Individual images shown from Video 2 (available) were taken at the indicated time points. (E) Lumen expansion in Caco-2 cells cultured as in D were treated with 0.1 μg/ml CTX, 100 μM of the PKA agonist 6-Bnz, 100 μM of the Epac agonist 8-Cpt, or 0.1 μg/ml CTX + 10 μM ouabain. Shown is the mean ± standard deviation for three independent experiments (50 cysts each). (F) Caco-2 was cultured for 10 d in three dimensions, fixed, and stained for DNA (blue), Na⁺/K⁺-ATPase (green), or CFTR (red). A single confocal section through the middle of the cyst is shown. Bars: (A–C) 25 μm; (D and F) 10 μm.